Gapdh

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GAPDH is a protein that functions as an enzyme involved in the glycolysis process, catalyzing the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. It is a common reference or housekeeping protein used in various assays and analyses.

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5 572 protocols using gapdh

Protein Expression Analysis in Cells

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Cells were lysed in RIPA buffer (Sigma‐Aldrich) supplemented with a protease inhibitor (Roche, Basel, Switzerland) and a phosphatase inhibitor (Roche). Protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA). Antibodies against PARP1 (#9542), cleaved PARP1 (#5625), caspase‐3 (#9665), cleaved caspase‐3 (#9664), cyclin D1 (#2978), p27 (#2552) and GAPDH (#2118) were purchased from Cell Signaling Technology (Cambridge, MA, USA). Isolated proteins were probed with the indicated primary antibodies followed by incubation with HRP‐linked secondary antibodies and detection using an ECL system (Thermo Fisher, USA). Protein expression levels were normalized to that of GAPDH (Cell Signaling Technology).

Weng W., Ni S., Wang Y., Xu M., Zhang Q., Yang Y., Wu Y., Xu Q., Qi P., Tan C., Huang D., Wei P., Huang Z., Ma Y., Zhang W., Sheng W, & Du X. (2017). PTTG3P promotes gastric tumour cell proliferation and invasion and is an indicator of poor prognosis. Journal of Cellular and Molecular Medicine, 21(12), 3360-3371.

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Protein Expression Analysis of Wound Healing

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Western blot (WB) analysis for proteins isolated from wound tissue of animals with or without CD34⁺ therapy was performed by following the standard procedures. Primary antibodies used were for MMP1, α-SMA (from Santa Cruz, CA), MMP3, MMP9, MMP13, SM22α (all from Abcam, USA), β actin, and GAPDH (both from Cell Signaling, Beverly, MA). Mouse, rabbit IgG-HRP conjugated (Cell Signaling, Beverly, MA) and goat IgG-HRP conjugated (Santa Cruz, CA) secondary Abs were used and specific bands were detected using enzyme-linked chemiluminescences (Pierce, IL). Densitometric analysis of developed bands was performed by using UN-SCAN-IT (gel 6.1 version) software. Relative density was calculated using respective GAPDH/ β-actin bands.
In a separate experiment, fibroblasts were cultured under serum deprived (1% FBS) conditions. Total protein was extracted in lysis buffer containing protease and phosphatase inhibitors from 5 different conditions of fibroblast cultures, such as added MG132 (10 µM), CD34⁺ cells, CD34⁺ cells plus MG132, or MG132 plus SP 600125 (JNK Inhibitor II, 20 µM) (from Calbiochem, Darmstadt, Germany), with medium alone serving as a control at both 6 h and 12 h time points. Twenty micrograms of total proteins were tested by WB analysis for levels of c-Jun and GAPDH (all from Cell Signaling, Beverly, MA) following the above-mentioned techniques.

Kanji S., Das M., Aggarwal R., Lu J., Joseph M., Pompili V.J, & Das H. (2014). Nanofiber-expanded human umbilical cord blood–derived CD34+ cell therapy accelerates cutaneous wound closure in NOD/SCID mice. Journal of Cellular and Molecular Medicine, 18(4), 685-697.

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Western Blot Analysis of Protein Expression

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Mouse lung tissues or cultured A549 cells were lysed with RIPA buffer (89900, Thermo Fisher Scientific, USA) containing 1 × protease inhibitor and phosphatase inhibitor (P1045, Beyotime, China). Protein concentrations were measured using the bicinchoninic acid assay (PH0326, Phygene). Equal amounts of proteins (25 or 30 μg) were subjected to gel electrophoresis and transferred onto polyvinylidene fluoridemembranes. These membranes were then blotted with FGF2 (1:1000 dilution, sc-74412, Santa Cruz) or GAPDH (1:1000 dilution, 5174, Cell Signaling Technology, Danvers, Massachusetts, USA) for mouse samples and FGF2 (1:1000 dilution, 20102, Cell Signaling Technology), phospho-p38MAPK (Thr180/Tyr182; 4511, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204; 4370, Cell Signaling Technology), p38MAPK (8690, Cell Signaling Technology), ERK1/2 (4695, Cell Signaling Technology), phospho-NF-κB p65 (Ser536; 3033, Cell Signaling Technology), phospho-IκBα (Ser32; 2859, Cell Signaling Technology), NF-κB p65 (8242, Cell Signaling Technology), or GAPDH (5174, Cell Signaling Technology) for A549 cell lysates. Subsequently, HRP-conjugated anti-rabbit (1:2000 dilution) or anti-mouse (1:3000 dilution) antibodies were used as secondary antibodies, and enhanced chemiluminescence (1705060, Bio-Rad) was conducted. Densitometry was performed using ImageJ (NIH, Bethesda, Maryland, USA) software.

Tan Y.Y., Zhou H.Q., Lin Y.J., Yi L.T., Chen Z.G., Cao Q.D., Guo Y.R., Wang Z.N., Chen S.D., Li Y., Wang D.Y., Qiao Y.K, & Yan Y. (2022). FGF2 is overexpressed in asthma and promotes airway inflammation through the FGFR/MAPK/NF-κB pathway in airway epithelial cells. Military Medical Research, 9, 7.

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Leonurine Promotes Osteogenic Differentiation

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The cells were then cultured with medium containing different concentrations of leonurine for 20 h and subsequently cultured with osteogenic induction medium for six days. The osteogenic-related mRNA level of OCN, OPN, Runx2 and the osteogenic protein level OPN, Runx2 expression level were further confirmed. OCN sense: 5′-TGAGGACCCTCTCTCTGCTC-3′, antisence: 5′-GGGCTCCAAGTCCATTGTT-3′; OPN sense: 5′-ATCTGAGTCCTTCACTG-3′, antisense: 5′-GGGATACTGTTCATCAGAAA-3′; Runx2 sense: 5’-GCACCCAGCCCATAATAGA-3’, antisense: 5’-TTGGAGCAAGGAGAACCC-3′; GAPDH sense: 5′-CAGGGCTGCCTTCTCTTGT-3′, antisense: 5′-TCCCGTTGATGACCAGCTTC-3′. GAPDH (1:2000) was purchased from Cell Signaling Technology (CST, Boston, MA, USA). OPG (1:500) and Runx2 (1:500) were purchased from Abcam (Abcam, Cambridge, UK).

Zhao B., Peng Q., Wang D., Zhou R., Wang R., Zhu Y, & Qi S. (2022). Leonurine Protects Bone Mesenchymal Stem Cells from Oxidative Stress by Activating Mitophagy through PI3K/Akt/mTOR Pathway. Cells, 11(11), 1724.

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Stretch-Induced Mechanotransduction in BMSCs

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At 0, 6, 12, and 24 h after stretch, whole-cell lysates were prepared from all three groups of cells, and the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using standard methods. Blots were incubated with primary antibodies (diluted to working concentrations of 1:1000 using blocking buffer) including rat monoclonal antibodies to ITGB1, ILK, RUNX2, and GAPDH (Cell Signaling Technology), overnight at 4 °C. After washing in TBS/T, secondary antibody, anti-mouse or rabbit immunoglobulin G conjugated to horseradish peroxidase (Biolegend, San Diego, CA, USA), was added for incubation at room temperature for 1 h. Immunoblot bands were visualized with an enhanced chemiluminescence solution (Millipore).
In addition, to confirm whether both FAK and FAK activation were involved in the stretch-mediated changes, we performed another group of western blot analysis to detect the protein expression of FAK, p-FAK and GAPDH (Cell Signaling Technology, Danvers, MA, USA) in BMSCs in the control group only at 0, 6, 12, and 24 h after exposed to mechanical stretch simulating distraction.

Hu P., Zhu X., Zhao C., Hu J., Luo E, & Ye B. (2019). Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch. Journal of Dental Sciences, 14(3), 225-233.

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Western Blot Analysis of EMT Markers

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Western blots were performed as described previously [29 (link)] using specific antibodies against PKM2, SOD2, E-cadherin (E-cad), Vimentin, members of the Snail family (Slug), extracellular signal-regulated kinase (ERK) 1/2, pERK1/2 and GAPDH (Cell Signaling Technology, MA, USA). GAPDH was used as a control (Cell Signaling Technology).

Wang W., He Q., Yan W., Sun J., Chen Z., Liu Z., Lu Z., Hou J., Shao Y., Zhou X, & Wang A. (2017). High glucose enhances the metastatic potential of tongue squamous cell carcinoma via the PKM2 pathway. Oncotarget, 8(67), 111770-111779.

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Western Blot Protein Analysis

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The protein (15–20 mg) extracted from cells was used for Western blot analysis. The antibodies utilized in this study included anti-YWHAZ (1:1000; Cell Signaling Technology, USA) and GAPDH (1:1000; Cell Signaling Technology, USA). GAPDH (1:2000; Cell Signaling Technology, USA) was used as a loading control.

Jia J., Sun J., Wang W, & Yong H. (2021). Long Noncoding RNA MLK7-AS1 Promotes Non-Small-Cell Lung Cancer Migration and Invasion via the miR-375-3p/YWHAZ Axis. Frontiers in Oncology, 11, 626036.

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Western Blot Analysis of HDAC9 Isoforms

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HASMCs and aortas were homogenized in RIPA buffer containing protease and phosphatase inhibitors (Sigma). Lysates (20 μg/lane) were mixed with denaturing buffer (1× Laemmli loading buffer with 10% of β-mercaptoethanol) and analyzed by SDS–PAGE/Western. Rabbit polyclonal anti-HDAC9 antibodies were used to detect the 125-kDa isoform of human HDAC9 in HASMCs (Origene, TA318928). Rabbit polyclonal antibodies directed against the murine short isoform of HDAC9 (MITR) were used to detect HDAC9 protein in mouse aortas (Abcam, #ab59718). Rabbit polyclonal antibodies directed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling #2118) were used to detect GAPDH protein. Blots were incubated with fluorescent-dye labeled anti-rabbit IgG IRDye 800CW (LI-COR, Lincoln, NE) and protein bands were imaged using a LI-COR Odyssey detection system (LI-COR, Lincoln, NE).

Malhotra R., Mauer A.C., Cardenas C.L., Guo X., Yao J., Zhang X., Wunderer F., Smith A.V., Wong Q., Pechlivanis S., Hwang S.J., Wang J., Lu L., Nicholson C.J., Shelton G., Buswell M.D., Barnes H.J., Sigurslid H.H., Slocum C., O’Rourke C., Rhee D.K., Bagchi A., Nigwekar S.U., Buys E.S., Campbell C.Y., Harris T., Budoff M., Criqui M.H., Rotter J.I., Johnson A.D., Song C., Franceschini N., Debette S., Hoffmann U., Kälsch H., Nöthen M.M., Sigurdsson S., Freedman B.I., Bowden D.W., Jöckel K.H., Moebus S., Erbel R., Feitosa M.F., Gudnason V., Thanassoulis G., Zapol W.M., Lindsay M.E., Bloch D.B., Post W.S, & O’Donnell C.J. (2019). HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype. Nature genetics, 51(11), 1580-1587.

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Western Blot Analysis of HDAC9 Isoforms

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Western Blotting Analysis of Protein Expression

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The cell lysate was collected after treatments for Wester blotting. The lysate samples were subjected onto 4–12% SDS-PAGE gel for electrophoresis as previously described [38 (link)]. Protein bands were then transblotted onto PVDF membrane (0.2 µm, ThermoFisher, USA), followed by blocking in 5% BSA-TBST buffer in room temperature for 1 h. The membrane was then incubated with primary antibodies targeting at: Akt (1:1000, #9272S, Cell Signaling Technology, MA, USA), p-Akt (1:1000, #9271S, Cell Signaling Technology, MA, USA), GSK-3β (1:1000, #9315S, Cell Signaling Technology, MA, USA), p-GSK-3β (Ser9, 1:1000, #5558S, Cell Signaling Technology, MA, USA), β-catenin (1:1000, # C2206-0.2ML, Sigma, USA), NeuroD1 (1:1000, #4373S, Cell Signaling Technology, MA, USA), GAPDH (1:1000, #2118 s, Cell Signaling Technology, MA, USA), overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies (Cell Signaling Technology, MA, USA), at room temperature for 1 h.. GAPDH (1:1000, #2118S, Cell Signaling Technology, MA, USA) was detected as a loading control. The antigen–antibody complexes were then detected with an ECL reagent kit (#10455145, Thermo Scientific). The protein analysis was performed with ImageJ software.

Liu Q., Telezhkin V., Jiang W., Gu Y., Wang Y., Hong W., Tian W., Yarova P., Zhang G., Lee S.M., Zhang P., Zhao M., Allen N.D., Hirsch E., Penninger J, & Song B. (2023). Electric field stimulation boosts neuronal differentiation of neural stem cells for spinal cord injury treatment via PI3K/Akt/GSK-3β/β-catenin activation. Cell & Bioscience, 13, 4.

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