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10 protocols using lf pa0212

1

Quantification of Cellular Metalloproteinases

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Western blotting analyses were conducted to detect proMMP-2 and -9 in cellular lysate using anti-MMP-2 (ALX-210-753, Enzo Life Sciences, Farmingdale, NY, USA) and anti-MMP-9 (3852S, Cell Signaling Technology, Danvers, MA, USA) antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as an internal loading standard, was detected using anti-GAPDH antibody (LF-PA0212, AbFrontier, Seoul, Korea). Cellular lysates were separated on 10% (w/v) SDS-PAGE and electrotransferred to PVDF membrane. After the blotted membrane was probed with primary antibodies overnight at 4 °C, it was incubated with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h at room temperature, and developed using an enhanced West-save up™ (AbFrontier, Seoul, Korea).
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2

Western Blot Protein Analysis

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Radioimmunoprecipitation assay buffer (Biosesang, Seoul, Republic of Korea), containing protease (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase (PhosSTOP; Roche, Basel, Switzerland) inhibitor cocktails, was used to isolate total cellular proteins. Protein concentrations are measured by BCA assay. Western blotting was conducted using standard protocols. 5% skim milk was used for blocking the nonspecific binding for 1 h. Primary antibodies were mouse anti- extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) antibody (sc514302, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-pERK1/2 antibody (sc136521, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-YAP antibody (sc-376830, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-pYAP antibody (PA5-17481, Invitrogen, 1:500 dilution) or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1:5000 dilution). A horseradish peroxidase (HRP) conjugated secondary antibody and a WEST-Queen™ Western Blot Detection Kit (iNtRON biotechnology, Seongnam, Republic of Korea) were used to detect immunoreactive bands. Data were quantified by video image analysis (Luminograph II, Atto, Tokyo). Protein bands were measured by Image J.
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3

Quantitative Analysis of MMP-2 and Nrf2 in Cell Lysates

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To quantitate proMMP-2 and nuclear factor erythroid 2-related factor 2 (Nrf2) in cellular lysates, western blotting analysis was performed using anti-MMP-2 (ALX-210-753, Enzo Life Sciences, Farmingdale, NY) and anti-Nrf2 (ab31163, Abcam, Cambridge, MA) antibodies as primary antibodies. GAPDH, used as an internal loading control, was detected using anti-GAPDH antibody (LF-PA0212, AbFrontier, Seoul, Korea). In brief, cellular lysates were separated on 10% (w/v) SDS-PAGE and electrotransferred to PVDF membranes. The blotted membrane was blocked with blocking buffer (2% BSA in 1× TBS-Tween 20), probed with primary antibodies overnight at 4 °C, incubated with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY) for 1 h at room temperature, and developed using an enhanced West-save upTM (AbFrontier, Seoul, Korea).
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4

Antibody Reagents for Cell Biology

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Primary antibodies against the following proteins were purchased [listed in the format “protein name (catalog number, supplier)”]: LC3B (NB100-2220, Novus; #3868, Cell Signaling Technology; or AM20212PU-N, ORIGENE), CNOT1 (14276-1-AP, Proteintech), CNOT7 (ab195587, Abcam), PRMT1 (07-404, Sigma-Aldrich), FLAG (A8592, Sigma-Aldrich; or F3165, Sigma-Aldrich), p62 (Cell Signaling Technology; 18420-1-AP), G3BP1 (13057-2-AP, Proteintech), GFP (sc-9996, Santa Cruz Biotechnology), U1 snRNP70 (sc-390899, Santa Cruz Biotechnology), β-actin (A5441, Sigma-Aldrich), and GAPDH (LF-PA0212, Ab Frontier).
The following secondary antibodies against the primary antibodies were used in this study: peroxidase-conjugated goat anti-mouse IgG antibody (AP124P, Sigma-Aldrich) and peroxidase-conjugated goat anti-rabbit IgG antibody (AP132P, Sigma-Aldrich) for western blotting, and Alexa Fluor ® 488-conjugated goat anti-mouse IgG antibody (A11017, Invitrogen) and rhodamine-conjugated goat anti-rabbit IgG antibody (31670, Invitrogen) for immunostaining.
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5

Western Blot Analysis of UPR Proteins

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The radioimmunoprecipitation assay buffer, (Biosesang, Seoul, Republic of Korea) including protease (Roche, Basel, Switzerland) and phosphatase (PhosSTOP; Roche) inhibitor cocktails, was employed to extract the total cellular proteins. Western blotting was performed using standard protocols. Nonspecific binding was blocked with 5% skim milk for 1 h. The primary antibodies were mouse anti-activating transcription factor 4 (ATF4; sc-390063, 1:500 dilution; Santa Cruz), anti-activating transcription factor 6 (ATF6; PA5-20215, 1:500 dilution; Thermo Fisher, Waltham, MA, USA), rabbit anti-XBP-1 antibody (sc-7160, Santa Cruz), mouse anti-LC3 (M186-3, 1:1000 dilution; MBL International Corporation, Woburn, MA, USA), or rabbit anti-GAPDH (LF-PA0212, 1:5000 dilution; AbFrontier Co., Ltd., Seoul, Republic of Korea). Horseradish peroxidase (HRP)-conjugated secondary antibody and a WEST-Queen™ Western Blot Detection Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) were employed to detect the immunoreactive bands.
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6

Protein Expression Analysis by Western Blot

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Western blotting analyses were performed to detect iNOS and filaggrin in cellular lysates using anti-iNOS (610332, BD Transduction Laboratories, KY, USA) and anti-filaggrin (SC-30229, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies, respectively. GAPDH, used as an internal loading standard, was detected using anti-GAPDH antibody (LF-PA0212, AbFrontier, Seoul, Korea). After the blotted membrane was incubated with primary antibodies overnight at 4°C, it was reacted with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h at room temperature and developed using an enhanced West-save up ™ (AbFrontier, Seoul, Korea).
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7

Western Blot Analysis of PTH, CaSR, and GAPDH

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The cultured cells were collected and lysed in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to the PVDF membrane (#ISEQ00010, Merck Millipore, Burlington, MA, USA). The following antibodies were used for Western blot analysis: mouse antibodies-PTH (1:500, #sc-69930, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CaSR (1:2000, #MA1-934, Invitrogen Waltham, MA, USA), and GAPDH (1:3000, #LF-PA0212, AbFrontier, Seoul, Korea).
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8

Evaluation of Cellular Signaling Pathways

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Radioimmunoprecipitation assay buffer (Biosesang, Seoul), supplemented with phosphatase (PhosSTOP; Roche, Basel) inhibitor cocktails and protease (Sigma-Aldrich, St. Louis, MO), was employed to obtain total proteins. SDS-PAGE electrophoresis and western blotting was conducted according to standard protocols. In brief, 5% skim milk was applied for inhibiting the nonspecific binding. The primary antibodies were as follows: rabbit anti- extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) antibody (ab17942, Abcam); rabbit anti- phospho-ERK1/2 (pERK1/2) antibody (ab4819, Abcam); mouse anti- SMAD2/3 antibody (sc-133098, Santa Cruz); rabbit anti-p38 antibody (sc-535, Santa Cruz); mouse anti-p-p38 antibody (sc-7973, Santa Cruz); rabbit anti-p63 antibody (ab124762, Abcam); mouse anti-YAP antibody (sc-376830, Santa Cruz); rabbit anti-caspase 3 antibody (sc-7148, Santa Cruz); mouse anti-caspase 9 antibody (sc-56076, Santa Cruz); mouse anti-LC3 antibody (M186-3, MBL); rabbit anti-PARP antibody (sc-9542, Santa Cruz); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier). After washing, an HRP-conjugated secondary antibody and a WEST-Queen™ western blot detection kit (iNtRON Biotechnology, Seongnam, Kyounggi-do) were applied for the detection of protein bands. Video image analysis (Luminograph II, Atto, Tokyo) was used to quantify the data [72 (link)].
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9

Protein Isolation and Western Blot Analysis

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Radioimmunoprecipitation assay buffer (Biosesang, Seoul, Republic of Korea) containing protease (11836153001, Complete Mini Protease Inhibitor Cocktail Tablets; Roche, Basel, Switzerland) and phosphatase inhibitor cocktails (04906837001, PhosSTOP; Roche) was used to isolate the total cellular proteins. Western blotting was performed using standard protocols. A 5% skim milk was used for blocking the non-specific binding for 1 h. Primary antibodies included the anti-ERK1/2 (sc-514302; 1∶500 dilution; Santa Cruz, CA, USA), rabbit anti-pERK1/2 (ab214362; 1∶500 dilution; Santa Cruz), mouse anti-microtubule-associated protein 1A/1B-light chain 3 (LC3, M186-3; 1∶1000 dilution; MBL), mouse anti-activating transcription factor 6 (ATF6, PA5-20215; 1∶500 dilution; Santa Cruz), or rabbit anti-GAPDH (LF-PA0212; 1∶5000 dilution; Abfrontier) antibodies. HRP-conjugated secondary antibody and a WEST-Queen™ Western Blot Detection Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) were used to detect the immunoreactive bands. The protein bands were quantified using the ImageJ Gel Analysis program.
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10

Western Blot Analysis of Cellular Signaling

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Cells were lysed for 30 min in RIPA buffer supplemented with protease (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase (PhosSTOP; Roche, Basel, Switzerland) inhibitor cocktails. Equal protein amounts of cell lysate were loaded on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, moved to polyvinylidene difluoride membranes and blocked with 5% skim milk in PBS/0.05% Tween 20 for 1 h. Primary antibodies for GAPDH (LF-PA0212, Abfrontier, Seoul, Korea), TCF4 (TCF7L2) (sc-13027, Santa Cruz Biotechnology, Dallas, TX, USA), β-catenin (ab325572, Abcam, Cambridge, MA, USA), cyclin dependent kinase 1 (CDK1) (ab131450, Abcam, Cambridge, MA, USA), cyclin D1 (sc-718, Santa Cruz Biotechnology, Dallas, TX, USA), SIRT1 (sc-15404, Santa Cruz Biotechnology), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (ab17942, Abcam), phospho-ERK1/2 (pERK1/2) (ab4819, Abcam), glycogen synthase kinase 3 beta (GSK3β, ab32391, Abcam), caspase-3 (sc-7148, Santa Cruz Biotechnology), notch1 (sc-376403, Santa Cruz Biotechnology), and Hes1 (sc-166410, Santa Cruz Biotechnology) were used. The immunoreactive bands were viewed using horseradish peroxidase-conjugated secondary antibodies (Bio-rad) and a WEST-Queen™ RTS Western Blot Detection Kit (iNtRON Biotechnology, Seongnam, Korea) and densitometric analysis was performed.
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