Mab2055

The total protein of A549 cells were harvested on following 24-hour incubation with ACPA (where maximum effect was observed) using RIPA buffer supplemented with phosphatase (cat#ab201113, Abcam) and protease (cat#78430, Thermo Scientific) inhibitors. Simple western assay was conducted following manufacturer’s guideline; 0.8 mg/ml (n = 4) of protein sample was separated on 12-230 kDa Wes Separation Module (cat#SM-W004, Protein Simple)^{41 (link)}. Nano-immunoassay was performed using primary anti-human antibodies for Akt (S473, S474, and S472) (cat#mab2055), JNK (T183/Y185 and T221/Y223) (cat#mab1387) and phospho-specific Akt (cat#mab887) and phospho-specific rabbit anti-human JNK (cat#mab1205) (all from R&D Systems). Total and phospho protein expression was assessed for Akt and JNK (Wes, Protein Simple, USA).

The RECs were seeded into 6-well plates and treated as described earlier. The cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors and phosphatase inhibitors. Proteins were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked with 5% skim milk. The blots were incubated with primary antibodies against E-cadherin (1:1000; ab1416; Abcam), vimentin (1:1000; ab92547; Abcam), AKT (MAB2055; R&D Systems, MN, USA), phosphorylated (p)AKT (1:1000; ab38449; Abcam), SMAD2/3 (AF3797; R&D Systems), pSMAD2/3 (1:1000; ab63399; Abcam), SNAIL/SLUG (1:1000; ab180714; Abcam), and β-tubulin (1:2000; ab15568; Abcam). The blots were incubated with horseradish peroxidase-conjugated secondary antibodies [anti-mouse (1:10,000; Abcam) and anti-rabbit (1:10,000; Sigma-Aldrich)] and visualized using an enhanced chemiluminescent system through gel documentation with an Amersham Imager 600. In addition to conventional Western blotting, automated protein separation and immunoblotting of the Western blots were performed using a Jess system (ProteinSimple, Bio-Techne, MN, USA). The proteins were normalized following the manufacturer’s instructions to normalize sample loading variability.