Rat umbilical cord mesenchymal stem cells (UCMSCs, CP-R302, Pricella, China), 293T cells (Pricella, China), DMEM medium (L110KJ, Yuanpei, China), Fetal bovine serum(AC03L055, life-ilab, China), GDF-6 (HY-P79333, MedChemexpress, USA), GDF-7 (92004ES10, Yisheng, China), CTGF (HY-P72154, MedChemexpress Biotechnology, USA), Goldenstar™ RT6 cDNA Synthesis Kit Ver.2 (TSK302M, Tsingke, China), qPCR Mix (SYBR Green I) kit and PCR primer (TSE002, Tsingke, China), RIPA lysis buffer (Beyotime, China), TNC (A1927, abclonal, China), MKX (ab236400, abcam, UK), SCX (ab58655, abcam, UK), GAPDH (A19056, abcam, UK), Hes1 (ab71559, abcam, UK), Hras (ab32417, abcam, UK), GAPDH Rabbit mAb (A19056, abclonal, China), Lentiviral Packaging Kit (41102ES10, Yisheng, China), Fetal bovine serum (26050070, Thermo, USA), trypsin (LP0042, Thermo, USA), PBS (10010001, Thermo, USA). Anti-CD34 antibody (PE), Anti-CD44 antibody (APC), Anti-CD45 antibody (APC-Cy7) Anti-integrin β1 (CD29)-FITC conjugate antibody were purchased from BD Pharmingen, USA.
Gapdh
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany, Canada, Hong Kong, Japan, Israel, Norway, Australia
About the product
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a key enzyme involved in the glycolytic pathway, catalyzing the reversible oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It plays a crucial role in cellular energy production.
Automatically generated - may contain errors
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Ripa buffer, by Beyotime (130 mentions)
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P akt, by Abcam (106 mentions)
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β catenin, by Abcam (87 mentions)
Polyvinylidene fluoride membrane, by Merck Group (86 mentions)
Cyclin d1, by Abcam (85 mentions)
Mmp 9, by Abcam (84 mentions)
Cleaved caspase 3, by Cell Signaling Technology (81 mentions)
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4 410 protocols using gapdh
1
Umbilical Cord Mesenchymal Stem Cell Characterization
2
Western Blot Analysis of Signaling Pathways
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Using RIPA buffer (Beyotime, P0013B) containing protease inhibitor (YEASEN, 16403140) and phosphatase inhibitor cocktail (YEASEN, 10226191), the trigeminal ganglia from mice and HEK293 cells were homogenized. Protein concentration was quantified using the BCA protein concentration assay, and loading buffer (YEASEN, 201315ES20) was added. The samples were then heated at 95°C for 5 min to denature the proteins. From each sample, 25 micrograms of protein were separated by SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride) membranes. After blocking with 5% non‐fat milk (Epizyme, PS112L) for 1 h, the membranes were incubated with the corresponding primary antibodies at 4°C for 16 h. A range of primary antibodies was utilized, including TRPC4 (1:1500; Alomone Labs, #ACC‐018 and Abclonal, A6996), p‐mTOR (1:1500; Abclonal, AP0115), mTOR (1:1500; Abclonal, A2445), p‐Akt (1:1500; Abclonal, AP1208), Akt (1:1500; Abclonal, A18675), p‐PI3K (1:1500; Cell Signaling Technology, 4292S), PI3K (1:1500; Abclonal, A4992), p‐ERK1/2 (1:1500; Abclonal, 83010S), ERK1/2 (1:1500; Abclonal, A4782), p‐P38 (1:1500; Abclonal, AP502), P38 (1:1500; Abclonal, A5049), p‐JNK (1:1500; Abclonal, AP1337), JNK (1:1500; Abclonal, A4867), p‐ATF2 (1:1500; Abclonal, AP1051), ATF2 (1:1500; Abclonal, A22718), and GAPDH (1:10000; Abcam, AB181602). The secondary antibody used was HRP goat anti‐Rabbit IgG (1:10000; Abclonal, AS070). Digital images were analyzed using densitometry with ImageJ software.
3
Protein Quantification and Immunoblotting
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Proteins were collected using RIPA reagent (Beyotime Biotechnology, Shanghai, China). Protein levels were quantified in cells and extracted using the BCA assay (Beyotime Biotechnology). Briefly, 20 μg of protein was separated by polyacrylamide gel electrophoresis (Beyotime Biotechnology) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime Biotechnology). After blocking for 2 h with 5% skim milk, the samples were incubated with primary and secondary antibodies (1:1000, CST, Danvers, MA, USA). Finally, the cells were observed using an enhanced chemiluminescence (ECL) assay kit (Beo Tianmei Biotechnology, Shanghai, China). The antibodies used were as follows: GPX4 (1:1000, Abcam, UK) and GAPDH (1:1000, Abcam).
4
Protein Extraction and Western Blot Analysis of Glioblastoma Cell Lines
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Glioblastoma cell lines U87 and SHG44 were obtained from ATCC (Manassas, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA), supplemented with 10% fetal bovine serum (FBS) and a penicillin/streptomycin antibiotic solution. Cultures were maintained in a humidified incubator at 37 °C with 5% CO2. To extract total proteins, cells were lysed using RIPA buffer (Beijing Biotime), containing phosphatase and protease inhibitors. Protein concentration was determined using a BCA protein assay kit. For Western blot analysis, 50 µg of protein from each sample was separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature. The membrane was incubated overnight at 4 °C with primary antibodies against GAPDH (Abcam, China) and NOD1 (Abcam, China). After primary antibody incubation, the membrane was washed and treated with HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized using the ChemiDoc XRS system.
5
Antibody Validation and Protein Regulation
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Mouse monoclonal antibodies against Flag (Anti-DDDDK-tag mAb, M185-3L, 1:1,000) were purchased from MBL Life Science; those against c-Myc (SC-40, 1:1,000), ubiquitin (sc-8017, 1:1,000), HA (sc-7392, 1:1,000), GAPDH (sc-32233, 1:1,000), USP11 (sc-365528, 1:1,000), COL1A1 (sc-293182, 1:500), vimentin (Abcam ab8978, 1:700), N-cadherin (Santa Cruz sc-8424, 1:1000), E-cadherin (Santa Cruz sc-21791, 1:1000), alpha-smooth muscle actin (α-SMA; Santa Cruz sc-53015, 1:500), ACTIN (Santa Cruz sc-47778, 1:5,000), and normal mouse IgG (sc-2025, 1:1,000) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against SFTPC (Cusabio CSB-PA021174GA01HU, 1:1000), SFTPC (Abcam ab90716, 1:500), USP19 (Proteintech 25768-1-AP, 1:1,000 ), Anti-Collagen 1 (Abcam ab21286, 1:500), α-SMA (Abcam ab124964, 1:500), OCT3/4 (ab18976; 1:500; Abcam Inc.), SOX2 (SC-365823, 1:250), SSEA4 (90231, 1:250; Millipore), fibronectin (CSB-PA13187C0Rb, 1:1,000), and 488/594-conjugated secondary antibodies (Cat. no. A21207 and Cat. no. A21203, 1:200; Life Technologies) were used.
Immunoprecipitation (IP) lysis buffer (Cat. no. 87787; Thermo Fisher), cell lysis buffer (Cat. no. R2002, Biosesang), protein 5X sample buffer (Cat. no. EBA-1052, Elpis Biotech), Protein A/G Plus agarose beads (sc-2003, Santa Cruz Biotechnology), protease inhibitor cocktail (Cat. no. 11836153001, Roche), the protein translation inhibitor cycloheximide (CHX; Cat. no. 239765, Merck), the proteasomal inhibitor MG132 (Cat. no. S2619, Selleckchem), the ubiquitin activating enzyme inhibitor MLN7243 (also called TAK243, Cat no. HY-100,487, Med Chem Express), puromycin (Cat. no. 12122530, Gibco), the USP11 inhibitor mitoxantrone dihydrochloride (MTX; Sigma-Aldrich, Cat no. M6545-10mg), the DUB inhibitor PR-619 (ab144641, Abcam), CCK-8 assay reagent (Dojindo Molecular Technologies, MD, USA), and 4', 6-diamidino-2-phenylindole (DAPI; Cat. no. H-1200, Vector Laboratories) were purchased and used.
Immunoprecipitation (IP) lysis buffer (Cat. no. 87787; Thermo Fisher), cell lysis buffer (Cat. no. R2002, Biosesang), protein 5X sample buffer (Cat. no. EBA-1052, Elpis Biotech), Protein A/G Plus agarose beads (sc-2003, Santa Cruz Biotechnology), protease inhibitor cocktail (Cat. no. 11836153001, Roche), the protein translation inhibitor cycloheximide (CHX; Cat. no. 239765, Merck), the proteasomal inhibitor MG132 (Cat. no. S2619, Selleckchem), the ubiquitin activating enzyme inhibitor MLN7243 (also called TAK243, Cat no. HY-100,487, Med Chem Express), puromycin (Cat. no. 12122530, Gibco), the USP11 inhibitor mitoxantrone dihydrochloride (MTX; Sigma-Aldrich, Cat no. M6545-10mg), the DUB inhibitor PR-619 (ab144641, Abcam), CCK-8 assay reagent (Dojindo Molecular Technologies, MD, USA), and 4', 6-diamidino-2-phenylindole (DAPI; Cat. no. H-1200, Vector Laboratories) were purchased and used.
6
Western Blot Analysis of Cyclin D1 and GAPDH
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Proteins from cells and tissues were isolated using RIPA buffer (P0013B; Beyotime Biotechnology, Beijing, China) supplemented with an EDTA-free protease inhibitor cocktail (04693159001; Roche Diagnostics, Basel, Switzerland) on ice for 15 min. The protein concentration was determined using a BCA assay kit (23227; Thermo Fisher Scientific, Inc. Waltham, MA, USA). The extracted proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then trans-blotted to polyvinylidene difluoride (PVDF) membranes (03010040001, Sigma, Darmstadt, Germany). Then the membranes were blocked with 5% skimmed milk in TBS with 0.05% Tween-20 for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies at a 1:1000 dilution at 4°C for 18 h. Next, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (ab181658; Abcam, Cambridge, UK) at a 1:5000 dilution for 1 h at room temperature. An enhanced chemiluminescence kit (407207; EMD Millipore, Darmstadt, Germany) was used to visualize the target proteins on the Tanon 4600 imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). The primary antibodies were Cyclin D1 (cat. no. 55506S; Cell Signaling Technology, Inc., Danvers, MA, USA), and GAPDH (no. ab128915; Abcam, Cambridge, UK).
7
Multiparametric Analysis of Organelle Function
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Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).
Secondary antibodies used for immunoblotting: DyLight800 mouse and rabbit (#SA5–10172 and #SA5–10044; Invitrogen), StarBright Blue 700 mouse and rabbit (#12004158 and #12004161; Bio-Rad), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (#115035003, #1:5,000; Jackson), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (#111035144, 1:5,000; Jackson). Fluorescent dyes used include MitoTracker Deep Red FM (#M22425; Thermo Fisher Scientific), MitoTracker Red CMXRos (#M7512; Thermo Fisher Scientific), LysoTracker Red DND-99 (# L7528; Thermo Fisher Scientific), and Hoechst 33342 (#H1399; Invitrogen).
Secondary antibodies used for immunoblotting: DyLight800 mouse and rabbit (#SA5–10172 and #SA5–10044; Invitrogen), StarBright Blue 700 mouse and rabbit (#12004158 and #12004161; Bio-Rad), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (#115035003, #1:5,000; Jackson), and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (#111035144, 1:5,000; Jackson). Fluorescent dyes used include MitoTracker Deep Red FM (#M22425; Thermo Fisher Scientific), MitoTracker Red CMXRos (#M7512; Thermo Fisher Scientific), LysoTracker Red DND-99 (# L7528; Thermo Fisher Scientific), and Hoechst 33342 (#H1399; Invitrogen).
8
Western Blot Analysis of Apoptosis Signaling
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Protease and phosphatase inhibitors from Roche were added to RIPA lysis buffer (from Beyotime Biotechnology) to obtain protein extracts from NP cells. A BCA Protein Assay Kit from Thermo Fisher Scientific was used to measure protein concentrations. Equal amounts of proteins were divided by SDS-PAGE, put on PVDF membranes (Millipore). After transfer, the membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature to reduce non-specific binding. The blocked membranes were incubated overnight at 4°C with primary antibodies against TGFBI, Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9, IKK, IKK, IKK, and p-IKB (Cell Signaling Technology) (1:1000, Abcam, USA) and GAPDH (1:5000, Abcam, USA). Protein bands were detected using an enhanced chemiluminescence system (Thermo Fisher Scientific) following incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Abcam, USA). As a loading control, GAPDH was utilized. The intensity of the bands was quantified using image analysis software and normalized to GAPDH to account for any variability in sample loading or transfer efficiency.
9
Quantifying Spinal Cord Protein Levels
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The total protein content of spinal cord tissues was isolated using radioimmunoprecipitation assay lysis buffer and then qualified using a BCA kit (Thermo Fisher, USA). Equal amounts of protein were loaded onto SDS–polyacrylamide gel and then transferred to polyvinylidene difluoride membranes membranes. Membranes were blocked with blocking solution (Epizyme Biotech, China) and incubated with primary antibody (Occludin, 1:2500, abmart, China, T55997; GAPDH, 1:10,000, abcam, USA, ab8245) overnight at 4 °C, followed by a 1 h incubation of secondary antibody. An enhanced chemiluminescence kit (Epizyme Biotech, China) was used to detect the protein signals. Protein was quantified using Image J software.
10
Detailed Reagents and Antibodies Protocol
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The reagents used in the study were obtained from various sources. G418 (4131) and neuregulin-1 (NRG1, 396HB) were purchased from R&D Systems. Doxycycline (DOX, D9891), cycloheximide (CHX, 01810), and MW167 (Calbiochem, 565755, a γ-secretase inhibitor) were acquired from Sigma‒Aldrich. EGF (PHG0311) and hygromycin B (106870110) were obtained from ThermoFisher Scientific. DAPT (2634, a γ-secretase inhibitor) was procured from TOCRIS, and VivoGlo™ Luciferin (P1043) was sourced from Promega. All reagents were used according to the manufacturer's instructions to ensure the accuracy and reproducibility of the experimental results.
The experiments utilized commercial antibodies obtained from various suppliers for Western blotting, immunofluorescence (IF) staining, immunoprecipitation (IP), and immunohistochemistry (IHC) staining, following the recommended protocols provided by the respective manufacturers. The antibodies listed below are specific for the following antigens: HNMT (H00003176-M12, Abnova; PA5-11,499, ThermoFisher Scientific), HER1 (8339, Cell Signaling), HER2 (3B5) (sc-284, Santa Cruz), trastuzumab (Roche), T-Dxd (Daiichi-Sankyo/AstraZeneca), HER3 (8339, Cell Signaling), HER4 (8339, Cell Signaling), α-Tubulin (GTX628802, GeneTex), pHER2 (Tyr1221/2) (2243, Cell Signaling), pHER2 (Tyr1248) (2247, Cell Signaling), p-AKT (4060, Cell Signaling), T-AKT (Santa Cruz, sc-265943), PS1 (5887, Cell Signaling), GAPDH (ab8245, Abcam), NuP98 (ab124980, Abcam), Lamin A/C (NB100-14457, Novus) and normal rabbit IgG (Sigma‒Aldrich). The experiment utilized a variety of secondary antibodies for different applications. Anti-rabbit IgG-HRP (GTX213110-01, GeneTex) and anti-mouse IgG-HRP (AP124P, Sigma‒Aldrich) antibodies were employed for western blotting. For IHC, anti-human HRP polymer (ab214883, Abcam), anti-mouse HRP polymer (VC001, R&D Systems), and anti-rabbit HRP polymer (VC003, R&D Systems) antibodies were used. Additionally, for IF staining, a range of antibodies from Jackson Immunological Research Laboratories were utilized, including anti-human FITC (109–096-003), anti-mouse rhodamine (715–025-151), anti-rabbit FITC (111–095-003), and anti-rabbit rhodamine (111–025-003) antibodies.
The experiments utilized commercial antibodies obtained from various suppliers for Western blotting, immunofluorescence (IF) staining, immunoprecipitation (IP), and immunohistochemistry (IHC) staining, following the recommended protocols provided by the respective manufacturers. The antibodies listed below are specific for the following antigens: HNMT (H00003176-M12, Abnova; PA5-11,499, ThermoFisher Scientific), HER1 (8339, Cell Signaling), HER2 (3B5) (sc-284, Santa Cruz), trastuzumab (Roche), T-Dxd (Daiichi-Sankyo/AstraZeneca), HER3 (8339, Cell Signaling), HER4 (8339, Cell Signaling), α-Tubulin (GTX628802, GeneTex), pHER2 (Tyr1221/2) (2243, Cell Signaling), pHER2 (Tyr1248) (2247, Cell Signaling), p-AKT (4060, Cell Signaling), T-AKT (Santa Cruz, sc-265943), PS1 (5887, Cell Signaling), GAPDH (ab8245, Abcam), NuP98 (ab124980, Abcam), Lamin A/C (NB100-14457, Novus) and normal rabbit IgG (Sigma‒Aldrich). The experiment utilized a variety of secondary antibodies for different applications. Anti-rabbit IgG-HRP (GTX213110-01, GeneTex) and anti-mouse IgG-HRP (AP124P, Sigma‒Aldrich) antibodies were employed for western blotting. For IHC, anti-human HRP polymer (ab214883, Abcam), anti-mouse HRP polymer (VC001, R&D Systems), and anti-rabbit HRP polymer (VC003, R&D Systems) antibodies were used. Additionally, for IF staining, a range of antibodies from Jackson Immunological Research Laboratories were utilized, including anti-human FITC (109–096-003), anti-mouse rhodamine (715–025-151), anti-rabbit FITC (111–095-003), and anti-rabbit rhodamine (111–025-003) antibodies.
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