Hrp conjugated goat anti rabbit igg

Rat corneal proteins were extracted with cold RIPA buffer. Aliquots having equal protein content were subjected to electrophoresis on 9% Tricine gels and then electrophorectially transferred to PVDF membranes. After 1 h blocking in 5% BSA, the blots were incubated with primary antibodies for: MK2(1:1000, Cell Signaling Technology, Danvers, MA, USA), Phospho-MK2(p-MK2, 1:1000, Cell Signaling Technology), p38(1:200, Santa Cruz Biotechnology Biotechnology, Dallas, TX, USA), Phospho-p38(p-p38, 1:1000, Cell Signaling Technology), PEDF(1:500, Abcam), VEGF(1:1000, Abcam) and HRP-conjugated anti-β-actin antibody (1:20,000, Sigma, Saint Louis, MO, USA) as a loading control. After washing each membrane three times with Tris-buffered saline containing 0.05% Tween 20 for 10 min, they were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, Sigma) or HRP-conjugated goat anti-mouse IgG (1:10,000, Sigma) for 1 h at room temperature. The specific bands were visualized by an enhanced chemiluminescence reagent (ECL, Lulong Inc, Xiamen, China), and the image intensity was calculated with a transilluminator (ChemiDoc XRS System; Bio-Rad, Philadelphia, PA, USA).

For immunodetection of the TGEV-N, p-EGFR, APN and TLR-6 proteins by Western blotting [20 (link)], rabbit anti-TGEV (VMRD, Hangzhou, China), rabbit anti-p-EGFR (CST) and rabbit anti-TLR6 (Bioss), followed by HRP–conjugated goat anti-mouse IgG and HRP–conjugated goat anti-rabbit IgG (Sigma) were used. The signal was detected using Super Signal West Pico lit (Thermo Scientific) and subjected to Image Reader LAS-4000 imaging system (FUJIFILM, Japan). The intensity of the bands in terms of density was measured and normalized against GAPDH expression. Three independent experiments and appropriate gel exposures yielded very similar results for each treatment modality.

Immunoblotting of lysate cells was carried out accordingly^{38 (link)}. Very briefly, PVDF membranes (PVDF; Biorad, Hercules, CA, USA) were incubated with primary antibodies (1:5000 dilution of mouse anti-GAPDH (Sigma-Aldrich, A2228)) and rabbit anti-NF-κB/P65 antibody (Abcam, ab90532, dilution: 1/500) that specifically recognizes phosphorylated NF-κB for 90 min. Subsequent to washing, membranes were incubated again with horseradish peroxidase (HRP)-conjugated goat antibody against mouse IgG (BBI, Boston Biomedical Inc., Cambridge, MA, USA) at a dilution of 1:16,000, or HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) at a dilution of 1:5000 for 45 min.

Proteins were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA-TBST (Sigma–Aldrich, St. Louis, MO, USA). Then, the membranes were treated with convalescent or immunized serum and HRP-conjugated goat anti-human IgG (H + L) (4A Biotech, Beijing, China) or HRP-conjugated goat anti-rabbit IgG (Sigma, Shanghai, China). Finally, the PVDF membrane was covered with ECL ultrasensitive chemiluminescence reagent (Beyotime, Jiangsu, China) and placed in a Bio–Rad gel imager for exposure and colour development. The recombinant S1 protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China), the unmutated spike RBD protein (SinoBiological Co., Ltd, Beijing, China), and the single-point mutation RBD protein (K417N, L452R, E484Q, N501Y, N439 K, and A520 V; SinoBiological Co., Ltd) were used in this study.

Lysates of nonadherent EAEC were prepared by suspending bacterial pellets in reducing SDS‐PAGE sample buffer. For adherent EAEC, infected T84 monolayers were lysed in ice‐cold lysis buffer (50 mM Hepes pH 7.4, 50 mM NaCl, 1% Triton X‐100) containing protease inhibitor cocktail (1:200, Sigma). After heat denaturation, proteins were separated in 15% SDS‐polyacrylamide gels and transferred to PVDF membranes (VWR) using a Mini‐PROTEAN Tetra Cell device (Bio‐Rad). Membranes were blocked in 5% skimmed milk in TBS/0.05% Tween‐20 for 1 hr and incubated in polyclonal rabbit anti‐dispersin (1:5,000; kindly provided by Christopher Icke, University of Birmingham) overnight at 4°C followed by HRP‐conjugated goat anti‐rabbit IgG (1:20,000; Sigma) for 30 min. Membranes were developed by enhanced chemiluminescence (Immobilon Western, Millipore) and imaged with a FluorChem E Imager (ProteinSimple). Densitometric analysis of band intensities was performed using ImageJ software (https://imagej.nih.gov/ij/).

Expression levels of wild-type and mutant GPs in transfected cells were verified by SDS-PAGE and Western blotting (Figure S1C). Thirty hours post transfection, cells were lysed with ice-cold RIPA lysis buffer [50 mM Tris buffer, pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS] supplemented with protease inhibitors (1.2 mg/ml leupeptin, 1 mM pepstatin A, 100 mM PMSF, and 1 mg/ml aprotinin). The cleared whole-cell lysate was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a 0.2-m nitrocellulose membrane using a semi-dry transfer apparatus (Thermo Fisher Scientific, Power Blotter- Semi-dry Transfer System). Membrane was blocked with Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBST) and 10% (w/v) bovine serum albumin. Membrane was incubated with biotinylated 3C10 anti GP antibody in 4°C overnight, washed with TBST, and then incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (#12-348 Sigma-Aldrich). For signal development, membrane washed with TBST before antibody binding was visualized using ECL reagent (#34579, Thermo Fisher Scientific).