Hrp conjugated anti rabbit and anti mouse igg antibodies

Ruscogenin (Rus, purity 99%; chemical formula: C27H42O4; molecular weight: 430.62) was purchased from J&K Scientific Ltd. (San Jose, CA, USA). Dextran sulfate sodium salt (DSS, molecular weight: 36,000–50,000) was purchased from MP Biomedicals (Irvine, CA, USA). Dulbecco’s modified eagle medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin was purchased from Invitrogen (Carlsbad, CA, USA). Overexpression plasmids for Flag-tagged TLR4 (pLV3-CMV-TLR4(human)-3×Flag-EF1a-CopGFP-Puro) and control plasmid were purchased from Miaoling Biology (Wuhan, China). The primary antibodies using in Western blotting were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Boston, MA, USA), and the HRP-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). The primary antibodies and the appropriate fluorescence conjugated secondary antibodies used in immunofluorescence staining were purchased from Abcam (Cambridge, UK).

Whole-cell lysate in radioimmunoprecipitation assay buffer, determination of protein concentration, SDS-PAGE, and immunoblotting were performed as described previously54 (link). For immunodetection, polyvinylidene fluoride membranes were blocked with 5% non-fat milk, incubated in Tris-buffered saline (pH 7.6; Sigma). The membranes were then incubated overnight at 4 °C with specific antibodies as follows: mouse monoclonal anti-AhR (sc-74571; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-p62/SQSTM1 (sc-28359; Santa Cruz Biotechnology); rabbit monoclonal anti-LC3B (GTX127375; Genetex, Irvine, CA, USA), anti-ATG12-5 (GTX124181; Genetex), anti-ATG7 (GTX61647; Genetex), and anti-BNIP3 (ab109362; Abcam, Cambridge, UK), rabbit polyclonal anti-E-cadherin (GTX100443; Genetex) anti-vimentin (GTX100619; Genetex); and mouse monoclonal anti-β-actin (A1978; Sigma). The blots were then incubated with HRP-conjugated anti-rabbit and anti-mouse IgG antibodies obtained from Cell Signaling Technology (7074 and 7076; Danvers, MA, USA). Enhanced chemiluminescence detection was performed according to the manufacturer’s protocol (Millipore, Billerica, MA, USA).

Protein levels were examined by western blot analysis. In brief, proteins lysed from cells were separated by SDS-electrophoresis and then transferred to a PVDF membrane. The membrane was blocked and incubated with primary antibodies. Antibodies against caprin-1 and G3BP1 were purchased from Proteintech (Wuhan, China). Antibodies against PCNA, pan-AKT, phospho-AKT Thr308, phospho-AKT Ser473, cyclin D1, mTOR, phospho-mTOR Ser2448, 4EBP1, and phospho-4EBP1 were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against EGFR, phospho-EGFR Tyr1068, RPS3, RPS6, RPL4, eIF4A, eIF4B, eIF4E, eIF4G, PABP, IRS1, and PDGFRA were purchased from Abcam (Cambridge, USA). HRP-conjugated anti-rabbit and anti-mouse IgG antibodies (Cell Signaling Technology, USA) were used as the secondary antibodies. Protein detection was performed with a chemiluminescence system (Bio-Rad, USA).