Mouse anti gapdh antibody

Macrophages from THP-1 differentiation were lysed with lysis buffer (Cell Signaling Technology, Beverly, MA), and the BCA assay kit (Beyotime, Jiangsu, China) was used to quantitate protein concentrations. Then, 40 μg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were then blocked and incubated overnight at 4°C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies for two hours. The ECL reagent (Pierce, Chester, UK) was used to detect the values of band intensities. ImageSaver (ATTO, Tokyo, Japan) was used for densitometry analysis. The primary antibodies used were rabbit anti-IFI44L antibody (Polyclonal, Abgent, San Diego, CA) and mouse anti-GAPDH antibody (Cell Signaling Technology).

The antibodies purchased were as follows: mouse anti-GAPDH antibody, rabbit anti-phospho-ERK1/2 and anti-phospho-AKT (Ser473) antibody from Cell Signaling Technology; anti-total ERK1/2 and anti-total AKT antibody from Santa Cruz Biotechnology; The protocol for immunoblotting has been described previously [13 (link)].

Isotretinoin was purchased from F. Hoffmann-La Roche Ltd (Basel, Switzerland). Metronidazole, doxycycline and rapeseed oil (Brassica rapa) were purchased from Sigma-Aldrich.
In order to investigate the purity of isolated CD4+CD62L+ T-cell fraction, cells were stained with antibodies specific against CD4 (APC-Cy7, GK1.5), CD62L (FITC, MEL-14), CD45RB (Pacific Blue, C363-16A) and CD127 (APC, A7R34). CD4+CD25+ T-cell fraction was stained with antibodies against CD4 (APC-Cy7, GK1.5), CD25 (PE, PC61.5) and Foxp3 (APC, FJK-16s). Antibodies were obtained from either BD Pharmingen (Basel, Switzerland) or eBioscience (San Diego, USA). Data was collected on a FACS Canto II (BD Biosciences, Allschwil, Switzerland) and analysed with FlowJo software (Ashville, USA).
Mouse anti–phospho-MAPK p38 (Thr¹⁸⁰/Tyr¹⁸²), rabbit anti-p38, rabbit anti–phospho-ERK1/2 (Tyr⁴²/Tyr⁴⁴), rabbit anti-ERK1/2 (p44/42 MAPK), rabbit anti-phospho-MSK1 (Thr⁵⁸¹), rabbit anti-MSK1, rabbit anti-SOCS3 and mouse anti-GAPDH antibodies were obtained from Cell Signaling Technology (Danvers, MA).

BHK-21 cells were infected with EMCV-C15 (m.o.i. of 1.0) and with EMCV HB10 as a positive control. At 48 h post-infection, cells were lysed in 5× SDS-PAGE loading buffer. The protein concentrations were determined with a bicinchoninic acid protein assay reagent (Pierce) according to the manufacturer’s instructions. An equal amount of each sample was loaded onto a 12% SDS-PAGE gel, followed by protein transfer to a PVDF membrane (Millipore). Each membrane was blocked for 1 h with blocking buffer (5% skimmed milk and 0.1% Tween-20 in PBS) and incubated with anti-VP1 primary antibody (1:1,000 dilution)^{19 (link)} or with mouse anti-GAPDH antibodies (Cell Signaling Technology, Inc.) to detect endogenous GAPDH, which served as a protein-loading control. Each membrane was washed three times with washing buffer (0.1% Tween-20 in PBS) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) for 1 h, after which the membranes were washed and exposed to the detection agent 3,3′-N-diaminobenzidine tertrahydrochloride (DAB, CWBIO, China). Protein sizes were determined by comparison with prestained protein ladders (Thermo Scientific, U.S.A.).

The rabbit anti-total Akt, anti-phospho-Akt(308), anti-phospho-Akt(473), anti-phospho-Smad2, anti-E-cadherin, anti-vimentin, anti-Snail1 and mouse anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). The mouse anti-Zeb1 antibody was ordered from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against rabbit IgG and mouse IgG conjugated with horseradish peroxidase were purchased from Abcam (Cambridge, MA). Fluorescent-labeled secondary antibodies were from Molecular Probes (Life Technologies, Carlsbad, CA). Common chemicals and reagents were purchased from Sigma-Aldrich unless otherwise noted. Plasmids encoding CIBN-GFP-CAAX, CIBN-CAAX, mCherry-CRY2-iSH2, CRY2-iSH2 and PH-Akt-mRFP were generated as described previously^{8 (link),9 (link)}.

Lung proteins were extracted with TNE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor (Complete Mini; Roche) using Microsmash (MS-100R; TOMY), 20 mM NaF, 2 mM Na₃VO₄), were sonicated and denatured with LDS sample buffer (Invitrogen) at 70°C. Proteins were electrophoresed on NuPAGE bis-tris precast gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Membranes were probed with anti-mouse ACE2 antibody [20 (link)] and anti-mouse GAPDH antibody (Cell Signaling TECHNOLOGY, 14C10). The blotting bands visualized with ECL reagent (Bio-Rad) using ChemiDoc Touch Imaging System (Bio-Rad). Image Lab software was used to quantify band intensity.