Protein sample preparation and western blot assay were performed as previously described (24 (link)). The primary antibodies used were: anti-MTMR3 (1:500; cat. no. 12443; Cell Signaling Technology, Inc.), anti-Cyclin D1 (1:1,000; cat. no. 60186-1-1g; ProteinTech Group, Inc.), anti-cyclin-dependent kinase 2 (1:1,000; CDK2; cat. no. 11026-2-AP; ProteinTech Group, Inc.), anti-p62 (1:1,000; cat. no. 19117-1-AP; ProteinTech Group, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-Cyclin E (1:1,000; cat. no. sc-247; Santa Cruz Biotechnology, Inc.), anti-Cyclin A (1:1,000; cat. no. sc-751; Santa Cruz Biotechnology, Inc.), anti-cell division cycle 25 A (1:1,000; cdc25A; sc-7157; Santa Cruz Biotechnology, Inc.), anti-microtubule associated protein 1 light chain 3 (LC3) A (1:500; cat. no. 4599; Cell Signaling Technology, Inc.), anti-LC3B (1:500; cat. no. 3868; Cell Signaling Technology, Inc.) and anti-GAPDH (1:20,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). Bound HRP-labeled secondary antibody (1:5,000; cat. no. SC-2005 or SC-2054; Santa Cruz Biotechnology, Inc.) was assayed by super ECL detection reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein density of western blots was analyzed using ImageJ 1.51k software (National Institutes of Health).
Anti p62
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
Anti-p62 is a laboratory antibody product used for detecting the p62 protein in biological samples. The p62 protein is involved in various cellular processes and can serve as a marker for certain conditions. The antibody is designed to specifically bind and identify the p62 protein, providing researchers with a tool for studying its expression and function.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Proteintech (9 mentions)
Anti beclin 1, by Proteintech (9 mentions)
Anti lc3b, by Proteintech (6 mentions)
Anti β actin, by Proteintech (6 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Anti bcl 2, by Proteintech (5 mentions)
Anti lc3, by Proteintech (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti lc3, by Cell Signaling Technology (3 mentions)
Sds page, by Bio-Rad (3 mentions)
Anti bax, by Proteintech (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti lc3, by Merck Group (2 mentions)
Anti lc3b, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti atg5, by Proteintech (2 mentions)
Bradford protein assay kit, by Beyotime (2 mentions)
Anti caspase 3, by Proteintech (2 mentions)
38 protocols using anti p62
1
Western Blot Analysis of Cell Signaling Proteins
2
Perfluorooctane Sulphonate (PFOS) Assay
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Perfluorooctane sulphonate (PFOS; potassium salt, purity ≥ 98%, Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich, Shanghai, China) and stored at 20°C. N-acetyl-cysteine (NAC), 3-methyladenine (3-MA), and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA; A7250-10G, M9281-100MG, and C6628-25G, respectively). Methyl thiazolyl tetrazolium (MTT) was purchased from Amresco (Solon, OH, USA, 0793-5g). A ROS assay kit was purchased from Beyotime Biotech (Nanjing, China, S0033). An AV-FITC/PI kit was purchased from KeyGen Biotech (Nanjing, China, #KGA108). Monodansyl cadaverine (MDC) and rhodamine 123 (Rh123) were purchased from Beyotime Biotech (P6659-20 μg and C2007, respectively). A BCA kit was purchased from Beyotime (Shanghai, China, P0012S). Anti-p62 was purchased from Proteintech (Shanghai, China, 55274-1-AP). Anti-LC3 was purchased from Sigma-Aldrich (Shanghai, China, L8918-200 μL). Anti-Bcl-2 and anti-Bax were purchased from CST (Shanghai, China, #3498 and #5023, respectively). Anticleaved-caspase-3 and β-actin were purchased from Santa Cruz, Paso Robles (CA, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies and the SuperSignal West Pico Kit were purchased from Thermo (Waltham, MA, USA).
3
Western Blot Analysis of Autophagy and Inflammation
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Protein samples were boiled for 5 min in 5× SDS-PAGE loading buffer and loaded onto 10–15% SDS polyacrylamide gels. Proteins were transferred to PVDF membranes, and membranes were blocked for 2 h in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 with 5% non-fat dry milk and then incubated at 4° C overnight with the following primary antibodies: anti-LC3B (1:2,000; Sigma-Aldrich), anti-P62 (1:5,000; Proteintech), anti-P65 (1:1,000; Cell Signaling Technology), anti-phospho-P65 (1:1,000; Cell Signaling Technology), anti-ICAM-1 (1:200; Santa Cruz Biotechnology), anti-PI3K (1:1,000; Cell Signaling Technology), anti-phospho-PI3K (1:1,000; Cell Signaling Technology), anti-Akt (1:1,000; Cell Signaling Technology), anti-phospho-Akt (1:1,000; Cell Signaling Technology), anti-phospho-p70S6K (1:1,000; Cell Signaling Technology), or anti-GAPDH(1:50,000; Proteintech). Blots were washed and then incubated with the HRP-conjugated secondary antibody (1:10,000; Proteintech), and blots were revealed using ECL. ImageJ software was used for the semi-quantification of protein expression.
4
Protein Analysis of OSCC and Normal Tissues
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The total protein was extracted using the kit (Beyotime Biotechnology, China), which included five samples of OSCC tissue paired with adjacent normal tissues. SDS-containing polyacrylamide gels were used to separate protein samples for transfer to polyvinylidene fluoride membranes (Millipore, USA). The membrane was treated with the primary antibody for an overnight period at 4 °C after being blocked with 5 percent BSA at room temperature. The primary antibodies were anti-p62 (1:1000; Proteintech, cat.no. 18420-1-AP), anti-LC3 (1:1000; Proteintech, cat.no. 14600-1-AP), anti-ATG5 (1:1000; Proteintech, cat.no. 10181-2-AP), anti-Bim (1:1000; Cell Signaling Technology, cat.no. #2933), anti-BCL-2 (1:1000; Proteintech, cat.no. 12789-1-AP), anti-Bax (1:1000; Cell Signaling Technology, cat.no. #14796), anti-GAPDH (1:2000; Elabscience, cat. no. E-AB-20072), and anti-Beclin-1 (1:1000; Proteintech, cat.no. 11306-1-AP). After three washes with TBST, the membrane was incubated for 1 h at room temperature with goat anti-rabbit IgG (1:10,000; Proteintech, cat.no. 10285-1-AP).
5
Protein Expression Analysis in HCC
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Tissue samples and HCC cells were lysed using RIPA lysis buffer supplemented with PMSF (1:100, G-CLONE) to obtain total proteins. The proteins concentration was estimated using the BCA protein quantification kit (Solarbio). Equal amounts of protein were subjected to SDS-PAGE, proteins were then transferred onto PVDF membranes (PerkinElmer). After blocking with skimmed milk or BSA, the membranes were incubated with the primary antibodies overnight at 4 ℃. Primary antibodies were as follows: anti-EVA1A (1:500, Abcam), anti-TP53 (1:1000, OriGene), anti-BAX(1:2000, OriGene), anti-BCL-2 (1:2000, Bioss), anti-p-JAK2 (1:1000, Cell Signaling Technology), anti-p-STAT3 (1:1000, Cell Signaling Technology), anti-MMP-9 (1:1000, Cell Signaling Technology), anti-LC3 (1:1000, MBL), anti-p62 (1:1000, Proteintech), anti-Beclin1 (1:2000, Proteintech) and anti-β-actin (1:1000, Servicebio). And then the blot was incubated with peroxidase-conjugated sheep anti-rabbit IgG (1:3000, Servicebio). Protein bands were detected using the ECL system. Each independent experiment was performed at least three times.
6
Succinic anhydride-conjugated PEI for P-glycoprotein silencing
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Succinic anhydride, b-PEI (MW 1,800), b-PEI (MW 250,000), 2-(7-Azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), N-ethyldiisopropylamine (DPIEA) et.al was purchased from Adamas-beta (Shanghai, China). All of solvent were purchased from Macklin (Shanghai, China). Chloroquine Phosphate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for Western Blotting and immunofluorescence including rabbit anti-LC3B, anti-p62, anti-Pgp, goat anti-rabbit IgG (H + L) (HRP, Cora Lite 594) were obtained from Proteintech (Wuhan, China). Alexa fluor 647-labeled goat anti-rabbit IgG (H + L), rabbit anti-CD44, anti-ki67, Ad-GFP-LC3B, Lyso-Tracker Red, TUNEL Apoptosis Assay Kit, acid phosphatase assay kit et.al were purchased from Beyotime (Shanghai, China). All other reagents for western blotting and gel electrophoresis were obtained from Solarbio (Beijing, China).
Targeting human P-gp siRNA sequences:
Sense: 5’-AAGAAGGAAAAGAAACCAACUdTdT-3’;
Anti-sense: 5’-AGUUGGUUUCUUUUCCUUCUUdTdT-3’.
All of siRNA were obtained by GenePharma Co. Ltd. (Shanghai, China).
Targeting human P-gp siRNA sequences:
Sense: 5’-AAGAAGGAAAAGAAACCAACUdTdT-3’;
Anti-sense: 5’-AGUUGGUUUCUUUUCCUUCUUdTdT-3’.
All of siRNA were obtained by GenePharma Co. Ltd. (Shanghai, China).
7
Western Blot Analysis of Protein Markers
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Proteins were exacted with an appropriate volume of lysis buffer. After electrophoresis on SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore). Antibodies used here were anti-LC3I/II (Sigma), anti-P62 (Proteintech), anti-notch1 (Cell Signaling Technology), anti-Hes-1 (Santa Cruz), anti-GAPDH (Cell Signaling Technology), anti-Tublin (Cell Signaling Technology) and goat anti-rabbit IgG HRP (Cell Signaling Technology).
8
Fluorescent Western Blotting for Autophagy Markers
9
Expressing Mutant VCP Proteins in U2OS Cells
10
Western blot and immunoprecipitation of brain proteins
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