Protein lysates were prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40 (Beyotime, ST366) with protease inhibitor cocktail (Cell Signaling Technology, 5871) and were used for an overnight immunoprecipitation (500 μg per sample) at 4°C with 10 μL of the following antibodies: anti-FLAG (GenScript, L00425); anti-TP53 (Santa Cruz Biotechnology, sc-47698); anti-HMGB1 (Santa Cruz Biotechnology, sc-26351) and anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402). A total of 20 μL of protein A/G Plus-agarose (Santa Cruz Biotechnology, sc-2003) was included in the incubation with the anti-TP53, anti-HMGB1 and anti-SQSTM1 antibodies. The resulting immunoprecipitates were analyzed using western blot assays.
Anti sqstm1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-SQSTM1 is a laboratory reagent used in scientific research. It is an antibody that specifically binds to the SQSTM1 protein, also known as p62. SQSTM1 is involved in various cellular processes, including autophagy and protein trafficking. This antibody can be used to detect and study the SQSTM1 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti hmgb1, by Santa Cruz Biotechnology (3 mentions)
Anti tp53, by Santa Cruz Biotechnology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Anti flag, by GenScript (1 mentions)
Np 40, by Beyotime (1 mentions)
Anti vimentin, by ABclonal (1 mentions)
Mg132, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti his tag, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti myc tag, by Cell Signaling Technology (1 mentions)
7 protocols using anti sqstm1
1
Immunoprecipitation and Western Blot Analysis
2
Immunohistochemical analysis of apoptosis and autophagy
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Tissue samples were deparaffinized with xylene, and an UltraSensitiveTM SP (Mouse/Rabbit) IHC kit (KIT-9730, MX Biotechnologies) was used for detection. After proteolytic digestion and peroxidase blocking, the tissue specimens were incubated with anti-Caspase-3 (#9664S, Cell Signaling Technology), anti-Cleaved Caspase-3 (#9662, Cell Signaling Technology), anti- SQSTM1 (sc-48402, Santa Cruz), anti-4E-BP1 (#9452, Cell Signaling Technology), anti-Vimentin (A19607, ABclonal), and anti-E-Cadherin (#3195T, Cell Signaling Technology) antibodies at a dilution of 1:200. The biotinylated secondary antibodies in the kit were used according to the manufacturer’s instructions after washing the samples. Then, the samples were washed with PBS three times and then incubated with streptavidin-horseradish peroxidase complex. A DAB substrate kit was used for staining. Liver tissues from control group mice were used as positive and negative control. Negative controls were prepared by substituting with normal rabbit or mouse IgG.19 (link) The immunoreactivity scores were calculated by two pathologists using a system based on multiplying the staining percentage (0: 0–5%, 1: 6–25%, 2: 26–50%, 3: 51–75% and 4: 76–100%) and intensity (0: negative, 1: weak, 2: moderate and 3: strong).
3
Phosphorylation-specific Beclin 1 Antibody Development
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Antibodies specific for Beclin 1 phosphorylated on Ser90 were generated by Abgent (San Diego, CA, USA, 1:500). Other primary antibodies used for western blotting were anti-Beclin 1 (Cell Signaling Technology, #3738, 1:1000), anti-GAPDH (KangChen, Shanghai, China, 1:10,000), anti-p-CaMKII (Cell Signaling Technology, #3361, 1:1000), anti-CaMKII (Santa Cruz, sc-1541, 1:500), anti-Bcl-2 (Santa Cruz, SC-7382, 1:500), anti-LC3 (Novus, Littleton, CO, USA, NB100-2220, 1:3000), anti-Id-1 (Santa Cruz, sc-488, 1:1000), anti-Id-2 (Cell Signaling Technology, #3431, 1:500), anti-SQSTM1 (Santa Cruz, sc-28359, 1:1000), anti-His-Tag (Cell Signaling Technology, #2366, 1:1000), anti-Myc-Tag (Cell Signaling Technology, #2276, 1:1000), anti-Flag (Sigma-Aldrich, F1804, 1:2000), anti-ubiquitin (Santa Cruz, sc-58449, 1:1000), anti-K63-linkage-specific polyubiquitin (Cell Signaling Technology, #5621, 1:1000), anti-TRAF-6 (Cell Signaling Technology, #8028, 1:1000), anti-GAP43 (Cell Signaling Technology, #8945 s, 1:1000), anti-NF68 (Cell Signaling Technology, #2837 s, 1:1000), anti-nestin (Santa Cruz, SC-23927, 1:1000), anti-vimentin (BD, 550513, 1:1000), and anti-E-cadherin (BD, 51-9001922, 1:1000). KN-93, MG132, and bafilomycin A1 were purchased from Sigma-Aldrich. Ionomycin was purchased from Cell Signaling Technology. EB1089 was purchased from Santa Cruz.
4
Antibody Sourcing for Cell Analysis
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Anti-β-actin (#A00702-100) antibody was purchased from GeneScript (Piscataway, NJ, USA), anti-Lamp-1 (#ab25630) was purchased from Abcam (Cambridge, MA, USA), anti-SQSTM1 (#sc-28359) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-cathepsin A (#AF1049) and anti-cathepsin D (#AF1014) were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against β-tubulin (#2128), Beclin1 (#3495), Caspase 3 (#9662), GAPDH (#2118), Lamin A/C (#4777), LC3A/B (#12741), PARP (#9542), and transcription factor EB (TFEB, #4240) were purchased from Cell Signaling Technology (Danvers, MA, USA).
5
Western Blot Analysis of Cellular Proteins
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Protein lysates (30–50 μg per sample) were separated on 8, 10 or 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Merck Millipore, IPVH00010). Incubations with primary and secondary antibodies and signal detection followed the manufacturer’s protocol using Western Lightning Plus-ECL (PerkinElmer, NEL105001EA). Quantitative image analysis was performed by using Quantity One software. The following antibodies were used: anti-GAPDH (Santa Cruz Biotechnology, sc-25778), anti-TP53 (Santa Cruz Biotechnology, sc-47698, sc-6243), anti-HMGB1 (Santa Cruz Biotechnology, sc-26351), anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402), anti-LC3B (Cell Signaling Technology, 2775s), anti-SQSTM1 (Cell Signaling Technology, 5114s), anti-HMGB1 (Cell Signaling Technology, 6893s), anti-ATG7 (Cell Signaling Technology, 2631s), anti-ATG5 (Cell Signaling Technology, 8540s), anti-cleaved-PARP (Cell Signaling Technology, 5625s), and HRP-labeled secondary antibodies (MultiSciences Biotech, GAR007, GAM007, and RAG007). The signal intensity was quantified using Quantity One® software and was shown to be within the linear range of detection.
6
Immunofluorescence Staining of Cellular Proteins
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Cells grown on poly-D-Lysine-coated cover slips were washed with phosphate-buffered saline (PBS; Gibco, 10010023) and fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS for 15 min at room temperature. They were then permeabilized with 0.2% Triton X-100 (BioFROXX, 1139ML100) in PBS for 15 min, blocked with nonfat dried milk in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) with Tween-20 (Aladdin, T104863) (TTBS) for 1 h and incubated with primary antibodies at room temperature for 2 h or 4°C overnight. After washing with PBS, the cells were incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies (Thermo Fisher Scientific, A21202, A10042, A10037, A11057) for 2 h, and then stained with DAPI (Dojindo, D212) for 5 min and mounted for fluorescence microscopy. The following primary antibodies were used: anti-TP53 (Santa Cruz Biotechnology, sc-47698), anti-HMGB1 (Santa Cruz Biotechnology, sc-26351), anti-SQSTM1 (Santa Cruz Biotechnology, sc-48402) and anti-LC3B (Cell Signaling Technology, 2775s).
7
Western Blot Analysis of CNP-Treated DU145 and PC3 Cells
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DU145 and PC3 cells were stimulated with 10 μg/mL CNPs for 24 h and then lysed in RIPA lysis buffer containing 1% PMSF and phosphatase inhibitors. Then, 10–25 μg of lysate proteins were separated by 10 or 12% SDS-PAGE and transferred to PVDF membranes (#ISEQ00010, Millipore). The membranes were blocked with 5% skim milk (#232100, BD Difco) in TBST at room temperature for 1 h and then incubated with anti-Caspase-3 (#9662, Cell Signaling Technology), anti-Cleaved Caspase-3 (#9664S, Cell Signaling Technology), anti-LC3 (NB100-2331, Novus Biologicals), anti-SQSTM1 (sc-48402, Santa Cruz), anti-4E-BP1 (#9452, Cell Signaling Technology), anti-Vimentin (#5741T, Cell Signaling Technology), anti-E-Cadherin (#3195T, Cell Signaling Technology), and Anti-β-actin antibodies (BM0627, Boster Biological Technology) at 4°C overnight. Subsequently, the membranes were incubated with an appropriate secondary antibody at room temperature for 1 h. Immobilon Western chemiluminescent HRP substrate was used to visualize the resulting protein bands. Three independent experiments were performed and repeated at least three times.
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