Mouse monoclonal antibody to gapdh

Infected HEK293 and AGS cell lines were harvested using cell scrapers, and heated at 95 °C for 5 min in 1× Laemmli buffer. Separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on gels with 6–10% polyacrylamide followed by Western blot analysis using ROTI®PVDF membranes (Carl Roth, Karlsruhe, Germany). The membranes were incubated for 1 h at 20 °C with TBS-T buffer (140 mM NaCl, 25 mM Tris–HCl pH 7.4 and 0.1% Tween-20) including either 3% BSA or 5% skim milk [45 (link)] to block non-specific binding sites. For detection, the following antibodies were used: mouse monoclonal antibody to GAPDH was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Phosphorylated and total CagA proteins were identified by successive probing of the blots with the mouse monoclonal α-pan-phosphotyrosine antibody PY99 (Santa Cruz Biotechnology) and rabbit polyclonal antibody against CagA (Austral Biologicals, San Ramon, USA) [46 (link)]. Polyvalent horseradish peroxidase (HRP)-coupled secondary goat antibodies were used to detect mouse and rabbit primary antibodies (Thermo Fisher Scientific, Massachusetts, USA). Subsequently, the blots were visualized using the ECL Prime chemiluminescence kit from GE Healthcare as described [47 (link), 48 (link)].

Restriction enzymes, DNA modifying enzymes, ProtoScript II First Strand cDNA Synthesis Kit, Q5 High Fidelity PCR Kit, endoglycosidase H (Endo H), and peptide-N-glycosidase F (PNGase F) were obtained from New England Biolabs (NEB) (Ipswich, MA, USA). Superscript II was obtained from Invitrogen (Carlsbad, CA, USA). TransIT-mRNA transfection reagent was obtained from Mirus Bio (Madison, WI, USA). Oligonucleotide primers and probes for DNA amplification and qRT-PCR were obtained from Sigma (St. Louis, MO, USA) and IDT (Coralville, IA, USA). mMESSAGE mMACHINE T7 ULTRA Transcription Kit was from Ambion (Austin, TX, USA). Anti-flavivirus monoclonal antibody D1-4G2-4-15 that reacts with ZIKV E protein was obtained from EMD Millipore (Billerica, MA, USA). Rabbit polyclonal anti-NS1 antibody was obtained from GeneTex (Irvine, CA, USA). Mouse monoclonal antibody to GAPDH was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies were obtained from Sigma (St. Louis, MO, USA) and Invitrogen (Carlsbad, CA, USA).

AP255 and AP1089, the affinity-purified rabbit polyclonal antibodies to CLIC4 and CLIC1, respectively, have been previously described [17 (link),32 (link)]. Commercial antibodies were as follows: Goat polyclonal antibody to mouse albumin, Bethyl Labs (Montgomery TX) #A90-134; rat monoclonal antibody to CD31 clone MEC13.3, Pharmingen (San Jose, CA) #550274; mouse monoclonal antibody to PCNA, Cell Signaling Technology (Danvers, MA) #2586; rabbit monoclonal antibody to Smad2/3, Cell Signaling Technology #8685; rabbit monoclonal antibody to phospho-Smad2/3, Sigma-Aldrich #SAB4504208; mouse monoclonal antibody to GAPDH, Santa Cruz Biotechnology (Santa Cruz, CA) #SC-32233; goat polyclonal antibody to CLIC5, Santa Cruz Biotechnology #SC-65041 Alexa Fluor 488 anti rat IgG, Life Technologies (Grand Island, NY); Cy5 goat anti rabbit IgG, Jackson Labs (Bar Harbor, ME) #111-175-144; FITC-lectin from Lotus tetragonolobus (LTA), Vector Labs (Burlingame CA) #FL-1321; Alexa Fluor 594-isolectin B4 from Griffonia simpicifolia (IB4), Life Technologies, #I21413; HRP-conjugated secondary antibodies, Thermo Scientific Pierce.