Phospho p44 42 mapk mab

Human pancreatic islets cultured with/without IGFBP3, with/without ecto-TMEM219, and in the presence/absence of TMEM219 siRNA were lysed and processed for ELISA (MyBiosource ELISA, MBS766157) to quantify Cleaved Caspase 8. Akt and phosphorylated-AKT, ERK1/2 and phosphorylated-ERK1/2 were detected by using the following antibodies (all from Cell Signaling Technology, Danvers, MA, USA): Phospho-AKT (Ser473, D9E) XP^® mAb (#4060) and panAKT (C67E7) mAb (#4691), Phospho-p44/42 MAPK mAb (Erk1/2, Thr202/Tyr204, #9101 S) and p44/42 MAPK mAb (Erk1/2, #9102 S), all used at 1:1000 concentration in TBS-T + 5% nonfat dry milk. Goat anti-Rabbit IgG (whole molecule) – Peroxidase antibody (1:5000, Merck, A0545) was used as a secondary antibody. The immunoblotting procedure is described in Supplementary Methods. Briefly, after incubation with appropriate primary and secondary antibodies, signals were revealed using enhanced chemiluminescence (Thermo Fisher Scientific) and visualized by a Uvitec Chemidoc mini hd9 imaging system (Uvitec, Cambridge, UK). Densitometric analysis was performed using Uvitec in-house software and expressed as mean ± SEM of data from at least three independent experiments.

Protein extraction, gel electrophoresis, and Western blotting procedures were carried out as described previously [4 (link)]. Primary antibodies (Cell Signaling Technology, Danvers, MA), along with their concentrations, were phospho-p38 MAPK antibody, #9211 (1:1,000); phospho-p44/42 MAPK mAb, #4370 (1:2,000 or 1:3,000); and phospho-eIF2α mAb, #3398 (1:1,000). The secondary antibody (1:20,000) was peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA).