Parental or shRNA Ca9-22 and OECM-1 cells were treated with or without 20 μM CQ for 1 h prior to LPLI treatment. The recovered LPLI-treated cell lysates were used to detect the accumulation of MAP1LC3-II, a lipidated and membrane-bound form of MAP1LC3, by immunoblotting to determine autophagic flux [21 (link)]. For immunoblotting, the cells were briefly rinsed with PBS (Biological Industries, Kibbutz Beit-Haemek, Israel) and lysed with an lysis buffer [1% Triton X100, 50 mM Tris HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA and a protease inhibitor cocktail (Roche Life Science)]. The proteins in the cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies against MAP1LC3 and ACTB (β-actin) (Sigma-Aldrich), SQSTM1 (BD Pharmingen), ATG7, RelA S536-P, and BECN1 (Cell Signaling Technology) overnight at 4°C. The proteins were probed with an HRP-labeled secondary antibody (Santa Cruz Biotechnology) and detected using an ECL reagent. The membranes were scanned and analyzed for protein expression level using a ChemiDoc XRS Imaging System (Bio-Rad Laboratories).
Sqstm1
Manufactured by BD
SQSTM1 is a laboratory equipment product offered by BD. It functions as a protein that plays a role in the process of autophagy, which is the degradation and recycling of cellular components. The core function of SQSTM1 is to facilitate the selective degradation of ubiquitinated proteins through the autophagy pathway. This product is designed for use in research and scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Becn1, by Cell Signaling Technology (1 mentions)
Rela s536 p, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Sartorius (1 mentions)
Hrp labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (1 mentions)
P crkl, by Cell Signaling Technology (1 mentions)
Tubb β tubulin, by Cell Signaling Technology (1 mentions)
Membrane integrity wb antibody cocktail, by Abcam (1 mentions)
A11122, by Merck Group (1 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A21429, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Rps6kb1, by Cell Signaling Technology (1 mentions)
P rps6kb1 t389, by Cell Signaling Technology (1 mentions)
Vdac1, by Abcam (1 mentions)
P mtors2448, by Cell Signaling Technology (1 mentions)
4 protocols using sqstm1
1
Autophagic Flux Measurement in Oral Cancer Cells
2
Western Blotting of Autophagy Signaling
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Western blotting was performed using antibodies against p-CRKL (Cell Signaling Technology, 3181), LC3B (Cell Signaling Technology, 2775) TUBB/β-tubulin (Cell Signaling Technology, 2146), SQSTM1 (BD Biosciences, 610833), ATG7 (Epitomics, 2054–1) and Membrane Integrity WB Antibody Cocktail (Abcam, ab110414). Primary antibody detection was by enhanced chemiluminescence (GE Healthcare/Amersham, RPN2106) using a horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, 7074 and 7076).
3
Antibody Validation for Protein Analysis
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The following commercial antibodies were used: rabbit polyclonal antibodies to GFP (Invitrogen, A11122), MYO1C (Sigma-Aldrich, HPA001768), EGFR (Santa Cruz Biotechnology, 1005 sc-03); mouse monoclonal antibodies to GFP (Abcam, ab 1218), LC3 (MBL International, M152–3), SQSTM1 (BD Biosciences, 610832), LAMP1 (Developmental Studies Hybridoma Bank, University of Iowa, H4A3-c), TUBA4A/tubulin (Sigma-Aldrich, T9026), CTSD/cathepsinD (BD Biosciences, 610800), FLOT1/flotillin 1 (BD Biosciences, 610820), FLOT2/flotillin2 (BD Biosciences, 610383), phospho-Thr389 RPS6KB/p70S6 kinase (Cell Signaling Technology, 9206), RPS6KB/p70S6 kinase (Cell Signaling Technology, 9202), phospho-Ser2448 MTOR (Cell Signaling Technology, 2971), MTOR (Cell Signaling Technology, 2972). Alexa Fluor 488- and 568-conjugated secondary antibodies were from Invitrogen (A21429 and A11029).
4
Western Blot Analysis of Autophagy Proteins
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Soluble proteins were harvested from cells by using SDS lysis buffer (50 mM Tris HCl, pH 6.8 containing 10% glycerol, 2% SDS, 10 mM dithiothreitol, 0.005% bromophenol blue). Equal volumes of proteins were separated by 8% or 12.5% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, IPVH00010). Blots were then blocked and immunolabeled overnight at 4C with primary antibodies, anti-MAP1LC3 (MBL International Corporation, PM036), p-MTOR (S2448) (Cell Signaling Technology, 2971), MTOR (Cell Signaling Technology, 4517), p-RPS6KB1 (T389) (Cell Signaling Technology, 9206), RPS6KB1 (Cell Signaling Technology, 9202), SQSTM1 (BD Biosciences, BD610833), TFEB (Cell Signaling Technology, 4240), VDAC1 (Abcam, 154,856), SLC6A4 (Abcam, 102,048), and ACTB (Abcam, 8226). Immunolabeling was visualized using an enhanced chemiluminescence kit (Bio-Rad Laboratories, 170–5061) according to the manufacturer’s instructions. Images were quantified using Image Lab software (Bio-Rad, Hercules, CA, USA). ACTB was used as an internal control. All band intensity is proportional the amount of target protein on the membrane with the linear range of detection.
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