Ab11304

At 24 h post transfection, the cells were collected and seeded onto a six-well plate. After 48 h, the cells were lysed and sonicated. Then, protein concentrations of lysates were measured using a Protein Assay Kit (Bio-Rad, Hercules, CA). The lysates were re-suspended in Laemmli buffer, denatured for 5 min at 98°C, and separated by Tris-glycine denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were blotted onto polyvinylidene fluoride membranes, blocked in 5% milk, and incubated overnight with the following primary antibodies: for endogenous protein detection, anti-E-cadherin (ab40772; Abcam, Cambridge, United Kingdom) or anti-α-tubulin (ab11304; Abcam); for MS2-22sTag protein detection, anti-HA (ab49969; Abcam) at a 1:1,000 (ab40772 and ab11304) or 1:2,000 (ab49969) dilution ratio in Can Get Signal Solution 1 (Toyobo) at 4°C. Subsequently, the proteins were incubated with the corresponding horseradish peroxidase–conjugated secondary antibodies (Thermo Fisher Scientific) at a 1:250 dilution ratio in Can Get Signal Solution 2 (Toyobo) for 1 h at room temperature. Chemiluminescent signals were generated using a SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) and captured on X-ray films (Fujifilm, Tokyo, Japan). The films were scanned, and signal intensities were quantified using ImageJ software.^*

Tissues homogenates were prepared as previously described [30 (link)]. Thirty μg of total protein was subject to SDS-PAGE and transferred onto PVDF membrane. Membranes were probed with the following primary antibodies: mouse anti-CypD (AbCam, ab110324), rabbit anti-ANT (AbCam, ab180715), rabbit anti-VDAC (CST, #4866), mouse anti-ATP Synthase (Abcam, ab5432), mouse anti-phospho-Akt S473 (CST, #4051), rabbit anti-Akt (CST, #4691), mouse α-Tubulin (Abcam, ab11304). Secondary antibodies were goat anti-mouse IR680 (LICOR) and goat anti-rabbit IR800 (LICOR). Membranes were scanned using the Odyssey infrared imaging system (LICOR).