Ab76247

HeLa and Caski cell lysates were prepared using RIPA buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in phosphate-buffered saline (PBS)] with freshly supplemented protease inhibitors cocktail (Roche). Next, the protein concentration was assayed by BCA (bicinchoninic acid) method, and 30 µg protein was separated on 10% SDS-PAGE membranes. Standard protocols were used for electrophoresis and immunoblotting analyses. Membranes were blocked with 5% milk in PBS. An anti-STK10 antibody (abcam, ab70484), anti-GAPDH rabbit polyclonal antibody (BBI, D110016), recombinant anti-ezrin antibody (abcam, ab40839), recombinant anti-moesin antibody (abcam, ab52490), recombinant anti-radixin antibody (abcam, ab52495) and Anti-ezrin (pThr567)/radixin (pThr564)/moesin (pThr558) antibody (abcam, ab76247) were used as the primary antibodies. The membranes were then incubated with IRDyeCW800-conjugated anti-rabbit immunoglobulin (LI-COR) and scanned with the LI-COR Odyssey imaging system (LI-COR).

Cells were fixed onto slides using 4% formaldehyde solution for 12 minutes at room temperature, washed with phosphate buffered saline (PBS) solution three times, and blocked with 5% bovine serum albumin (BSA) solution. Slides were then incubated with antibodies targeting LC3 (ab243506, Abcam), FUT4 (ab212396, Abcam), and p-ezrin (ab76247, Abcam) overnight at 4 °C. After washing in PBS solution, slides were incubated in the dark with the secondary antibodies (goat anti-rabbit IgG H&L (FITC), ab6717, Abcam; goat polyclonal secondary antibody to mouse IgG-H&L (Alexa Fluor^® 647), ab150115, Abcam) for 1–2 hours. The cell nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) and slides were mounted with the Prolong Gold Antifade Reagent (CST). The fluorescence intensities were evaluated by observation under fluorescence microscopy.