In immunofluorescence experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam, Cambridge, UK, cat # ab211324) 1:100; anti-SQSTM1/p62 (Abcam, cat # ab56416) 1:50; anti-β-Tubulin III (Merck Millipore, Burlington, MA, USA cat # T2200) 1:500; anti-MAP2 (1:500, Merck Millipore, cat # M9942) 1:500; anti-GFAP (Thermo Fisher Scientific, Waltham, MA, USA, cat # 13-0300), 1:800, donkey anti-rabbit-IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat # R37118) 1:1000; donkey anti-mouse-IgG Alexa Fluor 594 (Thermo Fisher Scientific, cat # A-21203, 1:1000); goat anti-mouse IgG1 CF 568 (Merck, cat # SAB4600313 1:1000); goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, cat # A21247, 1:1000); and DAPI (4′,6-diamidino-2-phenylindole Merck Millipore, cat #D9542) 1:5000. In Western blotting experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam cat # ab211324) 1:2000; anti-SQSTM1/p62 (Abcam cat # ab56416) 1:2000; Anti-GAPDH (Abcam cat # ab8245); 1:5000; anti-phospho-Akt (Ser473 D9E Cell Signaling, Danvers, MA, USA, cat #4060) 1:2000; anti-Akt (Cell Signaling, cat # 9272, 1:1000); goat anti-mouse IgG IRDye 800(Li-Cor, Lincoln, NE, USA, cat # 926-32210) 1:5000; and goat anti-rabbit-IgG Alexa 680 (Thermo Fisher Scientific cat # A21076) 1:5000.
Anti sqstm1 p62
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-SQSTM1/p62 is a lab equipment product that recognizes the SQSTM1/p62 protein. SQSTM1/p62 is a multifunctional protein involved in various cellular processes. This product can be used for research purposes to detect and study the SQSTM1/p62 protein.
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Lab products found in correlation
Anti lc3b, by Abcam (6 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti lc3b, by Cell Signaling Technology (4 mentions)
Anti β actin, by Proteintech (3 mentions)
Anti lc3, by Abcam (3 mentions)
Anti beclin 1, by Novus Biologicals (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti mtor, by Cell Signaling Technology (3 mentions)
Anti lc3, by Novus Biologicals (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti lc3b, by Novus Biologicals (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Anti axin1, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti gsk3β, by Cell Signaling Technology (2 mentions)
Anti p β catenin, by Cell Signaling Technology (2 mentions)
Anti ldha, by Cell Signaling Technology (2 mentions)
Anti histone h3, by Cell Signaling Technology (2 mentions)
42 protocols using anti sqstm1 p62
1
Immunofluorescence and Western Blot Antibody Protocol
2
Baicalin and 3-MA Modulate Autophagy and Apoptosis
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Baicalin and 3-MA were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). BAPTA-AM was purchased from Selleck Chemicals (Shanghai, China). Anti-Bax, anti-Bcl-xl, anti-cleaved caspase 3, anti-PARP, anti-mTOR, anti-p-mTOR, anti-AKT, anti-p-AKT, anti-cyclin D1, anti-cyclin B1, anti-cyclin A, anti-ZO-1, anti-Catenin, anti-Vimentin, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B, anti-MMP-2, and anti-beclin 1 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-HMGB1 and anti-p62/SQSTM1 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated and FITC-conjugated antirabbit or antimouse IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
3
Intracellular Staining of CD8+ T Cells
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A Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) was used for intracellular staining to permeabilize the cells. The permeabilization buffer of the kit will present at all times of the staining procedure. The sorted CD8+ T cells were incubated on ice with anti-LC3B (Abcam, Cambridge, MA, USA) and anti-p62/SQSTM1 (Abcam) antibodies for 15 min, and then incubation with corresponding secondary antibody for 15 min.
4
Intracellular p62/SQSTM1 and LC3B Staining
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Intracellular p62/SQSTM1 and LC3B staining was performed using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) to permeabilize the cells. After being washed, the cells were incubated with anti-p62/SQSTM1 (Abcam) and anti-LC3B (Abcam) antibodies on ice for 15 min, followed by incubation with secondary antibodies on ice for 15 min.
5
Western Blot Analysis of Autophagy Markers
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As previously mentioned48 (link), proteins were isolated and electroblotted. The membranes were blocked for 1 h with 5% BSA (ZSGB-BIO, Beijing, China) and incubated overnight at 4℃ with the primary antibodies (anti-Beclin-1, 1:1000dilution; anti-LC3, 1:2000 dilution; anti-RUNX2, 1:1000 dilution; anti-P62/SQSTM1, 1:5000 dilution, all from Abcam, MA, USA). After washing, membranes were incubated for 1 h with the secondary horseradish peroxidase antibody (Solarbio, Beijing, China). Finally, the intensity of the bands was determined using the Bio-Rad VersaDoc imaging system and the ECL kit (Solarbio, Beijing, China).
6
Immunohistochemical Analysis of Autophagy and Podocyte Markers
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The kidneys were immersed in 4% paraformaldehyde and embedded in paraffin. Kidney cross-sections were dewaxed heated for antigen retrieval, incubated with 3% H2O2 to block endogenous peroxidase activity and blocked with 1% BSA. Then, the sections were incubated at 4°C overnight with the following primary antibodies: anti-LC3 (B) (Cell Signaling Technology, 1:50), anti-P62/SQSTM1 (Abcam, 1:100), anti-nephrin (Abcam, 1:100), and anti-podocin (Abcam, 1:100). The sections were incubated with the relevant secondary antibodies, and the positive staining was visualized by staining with diaminobenzidine (DAB) and counterstaining with hematoxylin and examined under an electron microscope at a magnification of 400×. All images were quantified with Image-Pro Plus 6.0 software.
7
Autophagy and Apoptosis Pathway Analysis
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mASMCs were rinsed with ice-cold PBS once and then lysed in RIPA lysis buffer. The protein concentration of the sample was determined by Bio-rad ABS kit. 20 µg of the protein samples were loaded to each lane and separated in 12% SDS-PAGE gel, then blotted to PVDF membrane. The membrane was incubated overnight with anti-LC3B antibody (Novus; 1:1000 dilution) or anti-p62/SQSTM1 (1:1000; abcam, ab91526) for autophagy detection, or with anti-TRPM2 antibody (Abcam, ab11168, 1:1000 dilution) for expression study, or with anti-caspase-3 antibody (Cell Signaling Technology; 1:1000 dilution) for cell apoptosis detection, or with anti-ATP6V0A1 (Proteintech, 1:1000) for detection of vacuolar H+-ATPase. Immunodetection was accomplished with horseradish-conjugated secondary antibodies, followed by ECL detection system (Amersham Pharmacia).
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, China). An NE-PER™ Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, MA) was used according to the manufacturer’s instructions for extracting nuclear and cytoplasmic proteins from the different groups.
A 12% sodium dodecyl sulfate‐polyacrylamide gel was chosen for total protein separation, and the proteins were then transferred to nitrocellulose membranes (Millipore, USA). The membranes were incubated with primary antibodies, including anti-LC3B (Abcam Cat# ab192890, RRID: AB_2827794), anti-P62/SQSTM1 (Abcam Cat# ab207305, RRID: AB_2885112), anti-β-actin (Proteintech# 20536-1-AP), anti-LDHA (Cell Signaling Technology Cat# 3582, RRID: AB_2066887), anti-β-catenin (Cell Signaling Technology Cat# 8480), anti-p-β-catenin (Cell Signaling Technology Cat# 4176), anti-c-Myc (Covance Cat# MMS-150P-1000, RRID: AB_291322), anti-GSK3-β (Cell Signaling Technology Cat# 121456), anti-Axin1 (Cell Signaling Technology Cat# 2087, RRID: AB_2274550) and anti-Histone H3 (Cell Signaling Technology Cat# 4499, RRID: AB_10544537). Enhanced chemiluminescence reagents (Millipore, USA) were used to assess protein expression.
A 12% sodium dodecyl sulfate‐polyacrylamide gel was chosen for total protein separation, and the proteins were then transferred to nitrocellulose membranes (Millipore, USA). The membranes were incubated with primary antibodies, including anti-LC3B (Abcam Cat# ab192890, RRID: AB_2827794), anti-P62/SQSTM1 (Abcam Cat# ab207305, RRID: AB_2885112), anti-β-actin (Proteintech# 20536-1-AP), anti-LDHA (Cell Signaling Technology Cat# 3582, RRID: AB_2066887), anti-β-catenin (Cell Signaling Technology Cat# 8480), anti-p-β-catenin (Cell Signaling Technology Cat# 4176), anti-c-Myc (Covance Cat# MMS-150P-1000, RRID: AB_291322), anti-GSK3-β (Cell Signaling Technology Cat# 121456), anti-Axin1 (Cell Signaling Technology Cat# 2087, RRID: AB_2274550) and anti-Histone H3 (Cell Signaling Technology Cat# 4499, RRID: AB_10544537). Enhanced chemiluminescence reagents (Millipore, USA) were used to assess protein expression.
9
Immunoblotting Analysis of Cellular Proteins
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Total crypt cellular or nuclear extracts (30–50 μg protein/lane), were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane. The membranes were blocked with 5% BSA or 5% nonfat dried milk in Tris-buffered saline (TBS) (20 mM Tris-HCl and 137 mM NaCl, pH 7.5) for 1 h at room temperature (21 °C). Immunoantigenicity was detected by incubating the membranes overnight with the appropriate primary antibodies (0.5-1.0 μg/ml in 5% BSA or 5% nonfat dried milk) and Western blot analysis was performed as described [10 (link)]. Antibodies used were anti-DCLK1 (1:1000, ab31704, ab109029) from Abcam, anti-p62/SQSTM1 (1:400, H00008878-M01), anti-LC3B (1:300, NB100-2220) from Novus Biologicals, anti-GAPDH(1:3000), anti-β-actin (1:5000), from Sigma-Aldrich, anti-EZH2 (1:000), anti-H3 (1:1000) were from Cell Signaling Technology.
10
Antibody Sources and Reagents for Cell Signaling
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Anti-calreticulin (CRT) and anti-p62/SQSTM1 antibodies were purchased from Abcam. Anti-HN, anti-HSP90, and anti-STAT3 antibodies were obtained from Santa Cruz. Anti-β-actin and goat anti-rabbit antibody were purchased from Proteintech. Goat anti-mouse and Goat anti-rabbit antibodies for immunoblot analysis were obtained from Bioworld. The secondary antibodies of Alexa 488, Alexa 568 and Alexa 647 for immunofluorescence were obtained from Invitrogen. The following antibodies from Cell Signaling Technology were used: HMGB1, HSP70, poly (ADP-ribose) polymerase (PARP), p-eIF2α, p-STAT3 (Y705), Bcl-xl, Mcl-1, β-catenin. Mitoxantrone (MTX), Necrostain-1 (Nec-1), Z-VAD-FMK (Z-VAD), chloroquine (CQ), GSK2606414 (GSK), and C188-9 were obtained from Selleckchem. Recombinant interleukin-6 (IL-6) were obtained from PeproTech. Drugs were dissolved in dimethyl sulfoxide (DMSO) as stock solutions and stored at −20°C. ENLITEN®ATP Assay System Bioluminescence Detection Kit for ATP Measurement (#FF2000) was purchased from Promega. PI, Pierce®Protein Concentrator 2–6 mL/10K filters were purchased from Thermo Scientific. HMGB1 ELISA Kit II (#L534) was purchased from SHINO-TEST CORPORATION. Trypan blue dye was obtained from Sigma.
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