Rabbit polyclonal anti phospho eif2α

The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody (1:200; R&D Systems (Minneapolis, MN, USA), #BAM13361), mouse monoclonal anti-calnexin antibody (1:100; Novus Biologicals (Englewood, CO, USA), #NB300-518), rabbit polyclonal anti-GM130 antibody (1:200; Novus Biologicals, #NBP2-53420), guinea pig polyclonal anti-insulin antibody (1:50; GeneTex (Zeeland, MI, USA), #GTX27842). Secondary antibodies were from ThermoFisher Scientific: anti-streptavidin AlexaFluor-568 (1:200; #S11226), AlexaFluor-488 (1:200; #A11001 and #A11073) and AlexaFluor-568 (1:200; #A10042).
The antibodies used for immunoblotting were the following: mouse monoclonal anti-actin antibody (C4) (1:500), rat monoclonal anti-GRP78/Bip (1:250) and mouse monoclonal anti-vinculin (1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit polyclonal anti-Phospho-eIF2α (1:500), rabbit polyclonal anti-eIF2α (1:500) and mouse monoclonal anti-Chop (clone L63F7) (1:200) from Cell Signaling (Danvers, MA, USA), rabbit polyclonal anti-Atf4 (1:1000) from GeneTex. Secondary antibodies conjugated with HRP were from Biolegend (anti-mouse and anti-rabbit) and from Santa Cruz Biotechnology (anti-rat). Standard blocking conditions (5% milk in TBS-T) were used throughout, except when anti-P-eIF2α antibody was used, and 1% BSA in TBS-T was utilized.

Rabbit polyclonal anti-total PKR (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-phospho-PKR threonine 451 (Epitomics, Inc., Burlingame, CA, USA), rabbit polyclonal anti-eIF2α, rabbit polyclonal anti-phospho-eIF2α (Cell Signaling Technology, Inc., Danvers, MA, USA), and mouse monoclonal anti-β-actin antibody (Sigma-Aldrich) were used in the current study. Mouse monoclonal anti-NP mAb was generated using the NP of LaSota strain as an antigen expressed in a prokaryotic system. Briefly, sequences encoding the full length of NP gene was amplified by reverse transcription polymerase chain reaction and cloned into the bacterial expression vector pET32a (Novagen, Madison, WI, USA) in order to express the histidine-tagged protein heterologously in Eshcerichia coli strain BL21. The expression was induced by treatment with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 37°C for 4 h, and the expression product was purified using a His Band kit (Novagen) according to the manufacturer’s instructions. BALB/c mice were immuned by purified proteins for 4 times and sacrificed. The spleen of BALB/c mice was obtained for monoclonal antibodies preparation as previously
[39 (link)].