Hrp conjugated anti myc

Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40. Soluble proteins were immunoprecipitated using anti-G3BP1 (catalog no. 61559; Cell Signaling Technology), anti-G3BP2 (catalog no. 16276-1-AP; Proteintech), anti-Myc (catalog no. E6654; Sigma-Aldrich), anti-Flag (catalog no. A2220; Sigma-Aldrich), and anti-mouse IgG (catalog no. A0919; Sigma-Aldrich) antibodies. An aliquot of the total lysate (5% [vol/vol]) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-GFP (catalog no. SC-8334; Santa Cruz), HRP-conjugated anti-Myc (catalog no. SAB4200742; Sigma-Aldrich), HRP-conjugated anti-Flag (catalog no. A8592; Sigma-Aldrich), anti-G3BP1, anti-G3BP2, and anti-His (catalog no. SC-8036; Santa Cruz) antibodies. The antigen-antibody complexes were detected using an enhanced chemiluminescence (ECL) system (catalog no. 34095, Thermo Fisher). A PageRuler Western marker (catalog no. 6616, Thermo Fisher) was used as a molecular weight standard.

Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40 and protease inhibitor cocktail (Roche) (Cao et al., 2003 (link)). Soluble proteins were immunoprecipitated using anti-Flag (M2, Sigma), anti-Myc (Sigma), or IgG of the same isotype from the same species as a negative control (Sigma). An aliquot of the total lysate (5%, vol/vol) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-Myc (Sigma), HRP-conjugated anti-Flag (Sigma), HRP-conjugated anti-β-actin (Sigma), anti-VP35 (Creative Diagnostics), anti-IRF3 (Cell Signaling Technology), anti-phospho-IRF3 Ser396 (Cell Signaling Technology), anti-STING (Proteintech), or anti-NP (Sino Biological) antibodies. The antigen–antibody complexes were visualized via chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Millipore). A PageRuler Western marker (Thermo) was used as a molecular weight standard.

Cell lysates were prepared in prechilled Mammalian Protein Extraction Reagent (Thermo Scientific) containing protease inhibitor cocktail (Roche). Soluble proteins were immunoprecipitated using anti-Flag (Millipore, A2220) and anti-GFP (ABclonal, AE074) agarose beads or species-matched IgG of the same isotype as a negative control (Sigma-Aldrich, A0919 or A2909). An aliquot of the total lysate (5%, vol/vol) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma, A8592, 1:2,000 dilution), HRP-conjugated anti-Myc (Sigma, SAB4200742, 1:2,000 dilution), HRP-conjugated anti-GFP (Santa Cruz, sc-8334, 1:500 dilution), HRP-conjugated anti-GST (Invitrogen, MA4-004-HRP, 1:1,000 dilution), anti-PRKACA (BD Biosciences, 610980, 1:1,000 dilution), anti-CREB1 (Proteintech, 12208-1-AP, 1:1,000 dilution), and anti-Phospho-CREB (Ser133) antibodies. The antigen-antibody complexes were detected using an enhanced chemiluminescence system (GE Healthcare) and Immobilon Western HRP Substrate (Millipore, WBKLS0100). PageRuler Prestained Protein Ladder (Thermo) was used as a molecular weight standard.