Horseradish peroxidase hrp conjugated anti rabbit igg

HEK293T cells seeded in a 6-well cell culture cluster were cotransfected with porcine or human-Hsp90α/β-flag and SADS-CoV-N plasmids (Liu et al., 2021a ) or blank vector (as control) using Lipofectamine 3000 (Thermo Fisher). At 48 h post-transfection, cells were lysed with lysis buffer (25 mM Tris–HCl, 200 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, 25 mM β-glycerophosphate, 1% NP40, and protease cocktail [Biotool, Houston, TX]). 300 µL of supernatant was incubated with 4 µg of flag antibody overnight at 4 °C, followed by incubation with 30 µL of pre-washed Dynabeads™ Protein G at 4 °C for 12 h with mixing. After washing, the beads were eluted with 30 μL of elution buffer. The eluate was separated by 10% SDS PAGE and then applied to western blot analysis.
For cellular western blots, cells were lysed in lysis buffer, resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) overnight at 4 °C. Proteins were detected using the anti-myc antibody or anti-flag antibody at 1:1000 dilution, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000 dilution; Thermo Fisher Scientific).

The purified MCP protein, 293T cells transfected with pcDNA3.1-MCP recombinant plasmid and concentrated virus inoculum samples were separated by 12% SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) at room temperature for 2 h. Proteins were detected using the anti-MCP antibody at 1:1000 dilution followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000 dilution; Thermo Fisher Scientific).

Total cell lysate were prepared using RIPA buffer (Wako) supplemented with protease inhibitor cocktail (Roche Diagnostics). Then total cell lysate were mixed with sample buffer (Nacalai Tesque) and boiled at 95 °C for 5 min. Samples were then separated on a 4–20% Mini-PROTEAN TGX Gel (Bio-rad) at 200 V for 35 min. The separated proteins were transferred to a PVDF membrane using an iBlot system (Invitrogen). The membrane was blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO). The membrane was incubated with primary antibody. Antibody toward Phospho tyrosine 705 of STAT3 (#4113, Cell Signaling Technology, 1:2000), STAT3 (#12640; Cell Signaling Technology, 1:1000) and GAPDH (#2118; Cell Signaling Technology, 1:1000) were used. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Thermo Fisher Scientific) and anti-mouse IgG (SouthernBiotech) were used as a secondary antibody. Detection was carried out with SuperSignal West Femto Maximum Sensitivity substrate (Thermo scientific). Visualization of images was performed by a LAS1000 imaging system (GE Healthcare life sciences).

Antibodies against phospho‐Akt Ser⁴⁷³ (#9271), phospho‐Akt Thr³⁰⁸ (#9275), phospho‐TBC1D1 Thr⁵⁹⁰ (#6927), phospho‐JNK Thr¹⁸³/Tyr¹⁸⁵ (#9251), phospho‐p38 MAPK Thr¹⁸⁰/Tyr¹⁸² (#9216), phospho‐ERK1/2 Thr²⁰²/Tyr²⁰⁴ (#9101), total Akt (#9272), total TBC1D1 (#4629S), total‐JNK (#9252), total‐p38 MAPK (#9212), total‐ERK1/2 (#9102), total IκBα (#9242), and total‐acetyl CoA carboxylase (ACC, #3662) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho‐AS160 Thr⁶⁴² (#07‐802), phospho‐ACC Ser⁷⁹ (#07‐303), phospho‐TBC1D1 Ser²³⁷ (#07‐2268), and total AS160 (#07‐741) were from Millipore (Temecula, CA). Anti‐phospho‐AS160 Ser⁵⁸⁸ (#3028P2) was from Symansis Limited (Timaru, New Zealand). Anti‐GLUT4 antibody (#4670‐1704) was from Bio‐Rad AbD Serotec (Oxford, UK). Anti‐SPT2 antibody (ab23696) was from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG was from Biosource International (Camarillo, CA). HRP‐conjugated anti‐sheep IgG was from Millipore. HRP‐conjugated anti‐mouse IgG was from Santa Cruz Biotechnology. Enhanced chemiluminescence reagents (ECL, ECL plus, and ECL Prime) were obtained from GE Healthcare Life Sciences (Uppsala, Sweden). All other reagents were obtained from Sigma‐Aldrich (St. Louis, MO).