Cells were harvested and resuspended in lysis buffer (25 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitor cocktails) followed by centrifugation at 15,000 rpm for 20 min. Total proteins (40 μg) were loaded onto sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred to PVDF membrane. The membrane was probed with primary antibody for PPARγ (Cell Signaling Technology, Danvers, MA, USA), C/EBPα (Cell Signaling), cyclin D1 (Cell Signaling), cyclin B1 (Cell Signaling), cdk-4 (Abcam, Cambridge, UK), cdk-6 (Cell Signaling), LC3B (Cell Signaling), or SQSTM1/p62 (Sigma Aldrich). It was then incubated with HRP-conjugated anti-mouse (Santa Cruz, Dallas, TX, USA) or anti-rabbit IgG (Santa Cruz) secondary antibody. Immunoreactivities of proteins were visualized with an Enhanced Chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). Protein levels were quantified using the Fusion Solo system (Vilber Lourmat, Collegien, France). β-Actin (Sigma) served as a loading control.
P62 sqstm1
Manufactured by Merck Group
Sourced in United States, United Kingdom
P62/SQSTM1 is a protein that functions as a cargo receptor for selective autophagy. It binds to polyubiquitinated cargo and the autophagosome.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Polyacrylamide gel, by Bio-Rad (4 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Lc3a b, by Cell Signaling Technology (2 mentions)
Tom20, by Santa Cruz Biotechnology (2 mentions)
Tdp 43, by Proteintech (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Recombinant ifn γ, by R&D Systems (2 mentions)
γ h2ax clonejbw301, by Merck Group (2 mentions)
P62 sqstm1 gp62 c, by Progen Biotechnik (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (2 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (2 mentions)
23 protocols using p62 sqstm1
1
Western Blot Analysis of Adipocyte Markers
2
Western Blot Analysis of Autophagy Proteins
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Western blot analysis was performed as previously described9 (link), 10 (link) with antibodies against the following proteins: BECN1, 1:1000, (Abcam, Cambridge, UK Ab55878), β-actin 1:1000, (Cell Signaling Inc., Danvers, MA, USA, 4970), SQSTM1/p62 1:1000, (Sigma Aldrich, MABN130). The densitometric analysis was performed by Image J software and each data point was expressed as the mean±S.D. of independent experiments.
3
Western Blot Analysis of Autophagy Markers
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For western blot analysis, cells were harvested in 70 μL lysis buffer (3% SDS, 30 mM Tris base, pH 8.8, 5 mM EDTA, 30 mM NaF, 10% glycerol, 1 mM DTT) and 25–50 μg protein with 6x Laemmli buffer was loaded onto 4–20% Bio-Rad Mini- PROTEAN Precast Gels or 12% self-casted acrylamide/bisacrylamide (29:1) gels. After electrophoresis, the proteins were transferred to PVDF membranes with Bio-Rad Tris/Glycine buffer or self-made transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3) and stained with the following antibodies: ubiquitin – FK2 clone Millipore, 04–263; mCherry – Novus Biologicals, NBP1-96,752; GFP – Santa Cruz Biotechnology, sc-9996; LC3B – Cell Signaling Technology, 2775; SQSTM1/p62 – Sigma-Aldrich, P0067; ZFTVE1/DFCP1 – Cell Signaling Technology; WIPI2 – Invitrogen; ACTN2 (actinin alpha 2) – Sigma-Aldrich, A2543 or A7811; RPS6 (ribosomal protein S6) – Cell Signaling Technology, 2771; GAPDH – HyTest, 5G4. Western blots were either detected with LI-COR Biosciences Odyssey (Figures 4A, E, G, M and 5D ) or Biorad ChemiDoc (Figures 4C, I, J, K and 5E ).
4
Investigating Autophagy and Apoptosis Pathways
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Caspase-3 (#9662, 1:1000), Cleaved Caspase-3 (#9661, 1:1000), PARP (#9542, 1:1000), Bcl-xl (#2762, 1:1000), Bcl-2 (#2876, 1:1000), Bad (#9292, 1:1000), Bax (#2774, 1:1000), ATG-5 (#12994, 1:1000), ATG-7 (#8558, 1:1000), LC3A/B (#12741, 1:1000), Akt (#9272, 1:1000), p-Akt (Thr308) (#9275, 1:1000), p-mTOR (#2971, 1:1000), p-p70S6K (Thr389) (#9234, 1:1000) and β-actin (#4970, 1:1000) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). SRD5A1 (#66329-1-Ig, 1:1000) and p70S6K (#14485-1-AP, 1:2000) were purchased from Proteintech (Manchester, UK). The p-PI3K (Tyr607) (#AF3241, 1:1000) and mTOR (#AF6308, 1:1000) antibody were purchased from Affinity (Cambridge, UK). SQSTM1/p62 (#P0067, 1:1000) antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA) and goat Anti-rabbit IgG/Alexa Fluor 647 antibody (#bs-0295G-AF647, 1:500) was purchased from Bioss (Woburn, MA, USA). LY2940002 (#9901) were obtained from Cell Signaling Technology. Dutasteride (#A1659), hydroxychloroquine (HCQ) (#B4874) and 3-methyladenine (3-MA) (#A8353) were purchased from APExBIO (Houston, USA). Doxorubicin (#D1515) was purchased from Sigma-Aldrich.
5
Protein Expression Analysis of Autophagy Regulators
6
Oxidative Stress, Autophagy, and Apoptosis
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High‐glucose (25 mM) Dulbecco's modified Eagle's (DMEM‐H), foetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and Lipofectamine2000 were obtained from Thermo Fisher Scientific. Methyl viologen dichloride hydrate (paraquat, PQ), dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA), DAPI, acridine orange (AO), dansylcadaverine (MDC), and NAO dye were purchased from Sigma (MO, USA). Rapamycin (RAPA) and 3‐methyladenine (3‐MA) were purchased from Selleckchem. ST2825 was purchased from MedChemExpress. All molecular biological reagents were obtained from New England Biolabs. Primary antibodies against mTOR, phosphor(P)‐mTOR, P‐p53, Drp1, Tom20, cleaved‐caspase 3, and Bcl‐2 were purchased from Cell Signalling Technology. Anti‐Ki67, phosphorylated histone H2A.X (P‐γH2A.X), CD3, and CD68, and Alexa Fluor® 488‐conjugated or Alexa Fluor® 594‐conjugated second antibody were purchased from Abcam. Anti‐LC3B, SQSTM1/P62, and MyD88 were purchased from Sigma. Anti‐Beclin 1, CDK2, GPX1, 8‐OHdG, OPA1, and Fis1 were purchased from Bioss (Woburn, MA, USA). Anti‐SOD1, p53, PARP1, NDUFB8, and UQCRFS1 were purchased from Proteintech. Secondary antibodies horseradish peroxidase‐conjugated goat anti‐rabbit or mouse IgG was purchased from ZSGB‐Bio.
7
Cadmium-Induced Metabolic Dysfunction
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The following antibodies and chemicals were used: Cadmium chloride (CdCl2 ), antimicrotubule associated protein 1 light chain 3 betas (LC3B ), and SQSTM1/P62 (Sigma-Aldrich, St. Louis, MO). Melatonin (Shanghai, China). Horseradish peroxidase (HRP )-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-COX IV, anti-ACC, anti-FAS, anti-β-actin (Cell Signaling Technology, Danvers, MA). Anti-CPT1 and anti-PPAR-α (Proteintech, Wuhan, China). Dihydroethidium (DHE ), Oil Red O Staining Kit, and Hoechst 33258 (Beyotime Biotechnology, Shanghai, China). Micromitochondrial respiratory chain complex I–V activity assay kit (Solarbio, Beijing, China). Malondialdehyde (MDA ) assay kit, glutathione peroxidase (GSH-PX ) assay kit, superoxide dismutase (SOD ), triglyceride assay kit, and total cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing. China), and small interfering RNAs (siRNAs ) specific for PPAR-α (RiboBio Corporation, Guangzhou, China).
8
Macrophage Autophagy and Nitric Oxide Regulation
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Adherent macrophages were lysed using RIPA buffer (Sigma), and then
diluted in 2X Laemmli buffer, resolved using 4–20% polyacrylamide gels
(BioRad) transferred to PVDF membrane (BioRad) and detected with the following
antibodies: LC3b (Sigma L7543), p62/SQSTM1 (Sigma P0067), and GAPDH-HRP (sigma
G9295) or secondary goat-anti-Rabbit-HRP (Jackson 111–035-144). HRP was
detected using ECL (Biorad). For immunofluorescence (IF), adherent macrophages
were stimulated with 1 μg/mL bleomycin or unstimulated. Cells were fixed
and permeabilized before staining with antibodies against γ-H2AX (clone
JBW301, Millipore) and p62/SQSTM1 (GP62-C, Progen). For analyzing iNOS, adherent
macrophages were stimulated with 20 unit/mL recombinant IFN-γ (R&D
Systems) and 10ng/mL LPS (Sigma), After stimulation, media was removed and
replaced with cold PBS with 2 mM EDTA incubated on ice for 10 min to detach the
cells. Cells were first stained for live cells (Live/Dead Fixable Aqua,
Invitrogen) and surface staining with antibodies. Following by fixation and
permeabilization using BD Cytofix/Cytoperm (BD Biosciences), cells were stained
for iNOS (eBioscience 17–5920-82) and analyzed with flow cytometry.
diluted in 2X Laemmli buffer, resolved using 4–20% polyacrylamide gels
(BioRad) transferred to PVDF membrane (BioRad) and detected with the following
antibodies: LC3b (Sigma L7543), p62/SQSTM1 (Sigma P0067), and GAPDH-HRP (sigma
G9295) or secondary goat-anti-Rabbit-HRP (Jackson 111–035-144). HRP was
detected using ECL (Biorad). For immunofluorescence (IF), adherent macrophages
were stimulated with 1 μg/mL bleomycin or unstimulated. Cells were fixed
and permeabilized before staining with antibodies against γ-H2AX (clone
JBW301, Millipore) and p62/SQSTM1 (GP62-C, Progen). For analyzing iNOS, adherent
macrophages were stimulated with 20 unit/mL recombinant IFN-γ (R&D
Systems) and 10ng/mL LPS (Sigma), After stimulation, media was removed and
replaced with cold PBS with 2 mM EDTA incubated on ice for 10 min to detach the
cells. Cells were first stained for live cells (Live/Dead Fixable Aqua,
Invitrogen) and surface staining with antibodies. Following by fixation and
permeabilization using BD Cytofix/Cytoperm (BD Biosciences), cells were stained
for iNOS (eBioscience 17–5920-82) and analyzed with flow cytometry.
9
Visualizing Autophagy and Lipid Droplets
10
Western Blot and Immunostaining of α-Synuclein
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Protease inhibitor cocktail, phosphatase inhibitors, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO). Pam3CSK4 was purchased from InvivoGen (San Diego, CA). The following antibodies were used for western blot analysis and immunostaining: α-syn (Syn-1; BD Bioscience, San Diego, CA), Iba-1 (Wako, Richmond, VA), α-syn (Syn 211), p62SQSTM1, and β-actin (Sigma-Aldrich).
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