Total protein was extracted from PANC-1 cells using RIPA buffer (Beyotime Institute of Biotechnology) 72 h after transfection and 40 μg protein was separated by SDS-PAGE. Following electro-transfer of the proteins to a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane, the membrane was blocked using skim milk powder at room temperature (15–25°C) for 1.5 h. The membrane was then incubated at 4°C overnight with rabbit polyclonal BAX, Bcl-2 (Abcam, Cambridge, UK), phospho-p38, phospho-ERK1/2, phospho-JNK, phospho-MEK, p38, ERK1/2, JNK, and MEK (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies and mouse anti-human GAPDH monoclonal antibody (Beyotime Institute of Biotechnology), respectively. The membranes were then washed prior to incubation with secondary IgG antibody (Merck KGaA, Whitehouse Station, NJ, USA) labelled with alkaline phosphatase and visualized by ECL. The membranes were scanned and the relative level of protein expression was analyzed.
Mouse anti human gapdh monoclonal antibody
Manufactured by Beyotime
Sourced in United States
The Mouse anti-human GAPDH monoclonal antibody is a laboratory reagent used to detect and quantify the presence of the GAPDH protein in biological samples. GAPDH is a common housekeeping gene, often used as a reference protein in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Phospho mek, by Cell Signaling Technology (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Protein extraction kit, by Abcam (1 mentions)
Hrp labeled goat anti mouse igg, by Beyotime (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Te2000 inverted fluorescence microscope, by Nikon (1 mentions)
Horseradish peroxidase hrp labeled goat anti rabbit immunoglobulin ig g, by Beyotime (1 mentions)
One step rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Transwell chamber, by Corning (1 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (1 mentions)
Rabbit anti human pig3 polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
3 protocols using mouse anti human gapdh monoclonal antibody
1
Immunoblotting of PANC-1 Apoptosis Markers
2
Resveratrol Modulates MMP1/MMP13 in Chondrocytes
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Reagents in this study included resveratrol and collagen II (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin double-resistance (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), tetrazolium reagent (MTT; Sigma-Aldrich; Merck KGaA), rabbit anti-human MMP1 (cat no. 1973-1) and rabbit anti-human MMP13 (cat no. 1923-1) monoclonal antibodies (Epitomics, Burlingame, CA, USA), rabbit anti-human Sirt1 polyclonal antibody (cat no. ab110304; Abcam, Cambridge, MA, USA), protein extraction kit, bicinchoninic acid (BCA) protein assay kit, mouse anti-human GAPDH monoclonal antibody (cat no. AG019), horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (Ig) G (cat no. A0208), HRP-labeled goat anti-mouse IgG (cat no. A0216), ECL supersensitive luminescent liquid (Beyotime Institute of Biotechnology, Haimen, China), TRIzol reagent and one-step RT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). Transwell chambers (Corning Incorporated, Corning, NY, USA), ChemiDocTM XRS gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and TE2000 inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) were used.
3
Western Blot Analysis of Protein Signaling
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Protein extraction and western blot were performed according to published methods.18 The following primary antibodies were used: rabbit anti‐human PIG3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti‐human p‐FAK (Tyr397) monoclonal antibody, rabbit anti‐human p‐FAK (Tyr576) polyclonal antibody, rabbit anti‐human p‐FAK (Tyr925) polyclonal antibody, rabbit anti‐human total FAK monoclonal antibody, rabbit anti‐human p‐Src (Tyr416) monoclonal antibody, rabbit anti‐human total Src monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA) and mouse anti‐human GAPDH monoclonal antibody (Beyotime Institute of Biotechnology, Haimen, China) as the loading control. All primary antibodies were used at a 1000‐fold dilution.
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