α tubulin antibody

Total proteins from the cells were extracted using a RIPA Lysis and Extraction Buffer (Thermo Scientific™, Waltham, MA, USA) including Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Scientific™, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The PVDF membranes were incubated with primary antibodies at 4°C overnight, and then further incubated with secondary antibodies for 30 min on the following day. The immunoreactive signals were visualized using the chemiluminescence reagents WesternSure^® PREMIUM Chemiluminescent Substrate (LI-COR^®, Lincoln, NE, USA). Images were captured with the Odyssey^® XF Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The following antibodies were used: XIAP Antibody (Cell Signaling Technology, Danvers, MA, USA), α-Tubulin Antibody (Cell Signaling Technology, Danvers, MA, USA).

The protein was extracted from tissues or cells using a protein extraction kit (Shanghai Biyuntian Biotech), and the protein concentration was determined by BCA protein assay kit (Shanghai Biyuntian Biotech). 15–20 μg of total protein was mixed with 5 × SDS loading buffer and protease inhibitor, and then denatured by boiling at 100°C for 5 min. Proteins were separated by pre-electrophoresis at a constant voltage of 60 V for 20 min, followed by electrophoresis at a constant voltage of 100 V for 40–90 min according to the size of the target proteins. Separation gel was then transferred to the PVDF membrane at a constant voltage of 100 V for 90 min. The blots was washed three times with TBST, blocked with solution containing 5% BSA at room temperature for 1 h, and incubated with primary antibodies (AMPK antibody, phosphorylated AMPK antibody, PPARγ antibody, Cidec antibody, GAPDH antibody and α-tubulin antibody were purchased from Cell Signaling Technology) at 4°C overnight. After three times of wash in TBST, the blots were incubated with appropriate HRP-conjugated secondary antibodies. The chemiluminescence signals were developed using an ECL chromogenic solution (Advansta) and acquired by LAS-4000 gel imaging system.

Immunostaining was performed as described [24 (link)]. Briefly, cells were fixed with 4% PFA for 5 min and permeabilized using 0.1% Triton X-100 in PBS containing 1% bovine serum albumin (BSA) for 30 min at RT. Cells were incubated with α-tubulin antibody (Cell signaling #2125, Leiden, The Netherlands) or with a-RhoA (NewEast Biosciences; #26,904) at a dilution of 1:100 in PBS containing 0.02% Tween-20 (PBS-T) overnight at 4 °C, followed by secondary antibody incubation with Alexa Fluor 488 donkey anti-rabbit at a dilution of 1:200 (Abcam #150,073, Cambridge, UK) and rhodamine-conjugated phalloidin at a dilution of 1:100 (Invitrogen #10063052, MA, USA) at RT. After washes, images were stained with DAPI and visualized using a confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany).

Western blot was performed as described previously.⁴¹ (link) A cell fractionation kit (Cell Signaling Technology, USA) was used for nuclear fractionation. Antibodies against LGR6 and LGR4 were purchased from Proteintech, LGR5 and β-catenin from Invitrogen, and Bcl-2 and Bcl-xL from Cell Signaling Technology. α-tubulin antibody (Cell Signaling Technology) served as the loading control.