α tubulin monoclonal antibody

Cellular proteins were extracted with RIPA buffer (Sigma-Aldrich). Protein expression levels were examined by western blotting as previously described (Yang et al., 2016 ). Briefly, cells were lysed in lysis buffer and proteins were resolved by 4%–15% SDS-PAGE (Bio-Rad, 4561083). Proteins were then transferred to PVDF membranes (Bio-Rad, 1620177) followed by incubation with 5% milk in TBST blocking buffer for 1 h and incubation with antibody overnight. HRP-conjugated secondary antibody and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific 34094) were used to detect protein. SUCLG2 monoclonal antibody was purchased from Santa Cruz Biotechnology, Acetyl-α-tubulin (Lys40) monoclonal antibody was from Thermo Fisher Scientific, α-tubulin monoclonal antibody was from Cell signaling technology. β-Actin expression detected with anti-β-actin (Cell Signaling Technology, 8H10D10) served as the internal control.

Tissue lysates were analyzed by immunoprobing with custom-made rabbit polyclonal Ab3759 against the mouse CEACAM1 extracellular domain [24 (link)], and phosphorylated CEACAM1 (α-pCEACAM1) (Bethyl Laboratories, Montgomery, TX) [25 (link), 26 (link)]. Antibodies against phospho-insulin receptor β (pIRβ) (phospho-Y1361), IRβ (C18C4) (Abcam), FASN, phospho-Akt^Ser473 and Akt (Cell Signaling) were also used, α-Tubulin monoclonal antibody (Cell Signaling) was used for normalization. Blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody or anti-mouse IgG antibody (GE Healthcare Life Sciences, Amersham), and detected by ECL.