SC protein extracts were prepared by homogenization of mouse SC in extraction buffer [25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 2 mM MgCl2, 0.5 M urea and protease inhibitors (Sigma-Aldrich, P8340)] followed by centrifugation at 4°C for 20 min at 14 000 RPM. Supernatants only were used for western blotting. Protein extracts were resolved by SDS–PAGE and transferred to Hybond (Amersham) followed by detection with ECL reagent (Amersham). Antibodies included IFIH1 rabbit polyclonal antibody (1:3000, Proteintech Cat# 21775–1-AP), TRIM30 antibody (1:3000, Novus Biologicals, NBP2-41087), Anti-PCP4 antibody (1:5000, Abcam, ab197377), SQSTM1/p62 antibody (1:4000, Cell Signaling, Cat# 5114), mTOR antibody (1:4000, Cell Signaling, Cat# 2972), LC3B antibody (1:7000, Novus Biologicals, NB100-2220), GLT-1/SLC1A2/EAAT2 antibody (9 HCLC), ABfinity™ Rabbit Oligoclonal (1:3000, Thermo Fisher Scientific, Cat#: 711020) and monoclonal anti-β-Actin−peroxidase antibody, clone AC-15 (1:20 000, Sigma-Aldrich, A3854). The secondary antibody was peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (1:5000) (Jackson ImmunoResearch Laboratories, Cat# 111–035-144).
Sqstm1 p62 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The SQSTM1/p62 antibody is a laboratory tool used to detect the SQSTM1/p62 protein, which is involved in the autophagy process. This antibody can be used in various research applications, such as Western blotting, immunohistochemistry, and immunofluorescence, to study the expression and localization of the SQSTM1/p62 protein.
Automatically generated - may contain errors
Lab products found in correlation
Mtor antibody, by Cell Signaling Technology (3 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Affinipure goat anti rabbit igg h l antibody, by Jackson ImmunoResearch (1 mentions)
P8340, by Merck Group (1 mentions)
Dorsomorphin, by MedChemExpress (1 mentions)
Bafilomycin a1, by MedChemExpress (1 mentions)
Sch772984, by MedChemExpress (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Lc3a b d3u4c xp rabbit mab, by Cell Signaling Technology (1 mentions)
Z vad fmk, by Selleck Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Avertin, by Merck Group (1 mentions)
Fluphenazine, by Merck Group (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Sertraline, by Merck Group (1 mentions)
Rapamycin, by MedChemExpress (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Ampkα antibody, by Cell Signaling Technology (1 mentions)
Lc3b e5q2k mouse mab, by Cell Signaling Technology (1 mentions)
7 protocols using sqstm1 p62 antibody
1
Spinal Cord Protein Extraction and Western Blotting
2
Investigating Autophagy and Signaling Pathways
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Antibodies against mTOR (7C10) rabbit mAb (catalog 2983), phospho-mTOR (Ser2448) antibody (catalog 2971), p70 S6K antibody (catalog 9202), phospho-p70 S6K (Thr389) antibody (catalog 9205), AMPKα (D5A2) rabbit mAb (catalog 5831), phospho-AMPKα (Thr172) (40H9) rabbit mAb (catalog 2535), Atg5 (D5F5U) rabbit mAb (catalog 12994), Beclin 1 (D40C5) rabbit mAb (catalog 3495), SQSTM1/p62 antibody (catalog 5114), Akt antibody (catalog 9272), phospho-Akt (Ser473) antibody (catalog 9271), p44/42 MAPK (Erk1/2) antibody (catalog 9102), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) rabbit mAb (catalog 4377), and LC3A/B (D3U4C) XP rabbit mAb (catalog 12741) were obtained from Cell Signaling Technologies. Antibody against PARP (catalog CY6850) was obtained from Abways Technology. Antibody against β-actin (catalog A5441) was from MilliporeSigma. Sertraline (catalog S6319), fluphenazine (catalog PHR1792), erlotinib (catalog CDS022564), chloroquine phosphate (catalog PHR1258), avertin (catalog T48402, 152463), and dimethyl sulfoxide (catalog D2650) were purchased from MilliporeSigma. SCH772984 (catalog HY-50846), rapamycin (catalog HY-10219), dorsomorphin (catalog HY-13418A), and bafilomycin A1 (catalog HY-100558) were obtained from MedChemExpress, and 3-MA (catalog S2767) and Z-VAD-FMK (catalog S7023) were obtained from Selleck. All compounds were dissolved in dimethyl sulfoxide.
3
Western Blot Analysis of AMPK and mTOR
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The cells were lysed in RIPA buffer (Solarbio, R0010). The protein concentration was determined using the BCA Protein Assay Kit (Meilunbio, MA0082). Equivalent amounts of protein were separated through SDS-polyacrylamide gels and electroblotted onto PVDF membranes (Bio-Rad, 1620177). After blocking using a 5% BSA Blocking Buffer (Solarbio, SW3015), the membranes were incubated with the primary antibody at 4 °C overnight. The antibodies used were AMPKα Antibody (Cell Signaling Technology, 2532, Danvers, MA, USA), Phospho-AMPKα Rabbit mAb (Cell Signaling Technology, 2535), mTOR Antibody (Cell Signaling Technology, 2972), Phospho-mTOR Antibody (Cell Signaling Technology, 2971), LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, 83506), SQSTM1/p62 Antibody (Cell Signaling Technology, 5114), and β-Actin Antibody (Cell Signaling Technology, 4967). Finally, the relevant protein was visualized through staining with an appropriate HRP-conjugated secondary antibody for 1 h and then enhanced with chemiluminescence. The expression of the protein was quantified using ImageJ software.
4
Immunoblotting Analysis of Autophagy
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Cultured cells were extracted with RIPA buffer containing protease inhibitors and a phosphatase inhibitor cocktail (Roche). The protein concentration was determined by the BCA assay (Pierce). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore) and analyzed by immunoblotting. The antibodies used were as follows: LC3A/B antibody (#4108, 1:2000) and SQSTM1/p62 antibody (#5114, 1:2000) were purchased from Cell Signaling Technology, and β-actin (#A5316, 1:2500) was purchased from GenScript.
5
Western Blot Analysis of Autophagy and Inflammatory Markers
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The tissue samples from the frozen specimens were dissected and homogenized in lysis buffer with a Dounce homogenizer. Samples were lysed using radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysed tissues were centrifuged at 10,000 × g for 20 minutes for clarification. After quantification of the protein content, the lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12%-15% acrylamide gels. Immunoblotting was conducted after transferring the lysate to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The corresponding antibodies were as follows: anti-rabbit polyclonal atg8/LC3 antibody, SQSTM1/p62 antibody, and Beclin 1 antibody, obtained from Cell Signaling (Irvine, CA); phospho-JNK, p38, and p65, obtained from Cell Signaling (Danvers, MA); interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and β-actin (sc-70,319), purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence Western Blotting Detection Reagents (Millipore) were used for the detection of bands, and the quantification of blots was conducted with ImageJ densitometry software (National Institutes of Health, Bethesda, MD) for densitometric analyses.
6
Molecular Signaling Pathway Analysis
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Galectin‐3 antibody, SAPK/JNK antibody, phospho‐SAPK/JNK (Thr183/Tyr185) antibody, Phospho‐PI3 Kinase Class III (Ser249) antibody, PI3 Kinase Class III antibody, SQSTM1/p62 antibody, LC3B antibody, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody were purchased from Cell Signaling (Danvers, MA, USA). Collagen type 1A1 (COL1A1) antibody and interleukin (IL)‐1R‐associated kinase 1 (IRAK‐1) were purchased from Santa Cruz (Dallas, TX, USA). The α‐SMA antibody was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Alexa Fluor 488 goat anti‐mouse immunoglobulin G (IgG) and Alexa Fluor 594 goat anti‐rabbit immunoglobulin G were purchased from Invitrogen (Carlsbad, CA, USA). DOX hydrochloride and SP600125 were purchased from Sigma‐Aldrich. Chloroquine was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Pioglitazone hydrochloride was purchased from Fujifilm (Tokyo, Japan). ODN2088 and GW1929 were purchased from AdipoGen Life Sciences (San Diego, CA, USA).
7
Western Blotting Antibody Specifications
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