Western blot analysis of cytoplasmic markers

Proteins extracted from Hela cells were used as cellular control, the pellet was resuspended in RIPA buffer (50 mM Tris–HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 0.1 mM CaCl₂, and 0.01 mM MgCl₂ supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), incubate 30 min in ice vortexing every 10 min and centrifuge 20 min at 20,000 × g. An equal volume of bioproducts (38 µL) was loaded and separated on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad, USA). Bioproducts and cell lysates were treated with protein loading dye (Laemmli sample buffer; Bio-Rad) with freshly added β-mercaptoethanol 10%; v/v; Sigma, Germany) and boiled for 5 min at 95 ℃ before SDS-PAGE. Proteins were subsequently blotted to a nitrocellulose blotting membrane (0.2 µm; 1,060,000; GE Healthcare, USA). Membranes were blocked in 5% BSA (Carl Roth, Germany) in 0.1% Tween in TBS (TBS-T). After blocking, blots were probed with the following primary antibodies diluted in 5% BSA/TBS-T: Calnexin (1:500 dilution, E-10, Santa Cruz Biotechnology). After overnight incubation at 4 ℃, membranes were washed 3 times with TBS-T and subsequently incubated with the secondary antibody dilution: Polyclonal Goat anti-mouse HRP (1:5000 dilution; P0447) for 1 h at room temperature followed by washing. Blots were then developed using Western Bright ECL (541,004; Biozym Scientific, Germany) and protein bands were detected using the FusionCapt Advanced Solo 4 (Vilber, Germany).