The same blot was reprobed with an anti-Na+/K+-ATPase antibody (1 1000, Abcam, Cambridge, MA, USA) as a loading control after peeled the antibodies off by ReBlot Plus Strong (Merck, Darmstadt, Germany).
Rabbit anti-LRRC25ic polyclonal antibody (1 μg/mL) was used to detect LRRC25, β-actin was detected with a mouse anti-human β-actin (1 3,000 dilution, Clone AC-74, Sigma, USA) as a loading control, and secondary antibodies were from Cell Signaling Technology (USA), including HRP-conjugated goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody (1 5,000 dilution).
We used an antibody directed against γ-tubulin (Santa Cruz, USA) as a loading control.
Membranes were stripped and re-probed for β-actin (1 : 1000, Cell Signalling Technology Inc., Danvers, MA, USA) as a loading control.
The membranes were stripped and re-blotted with an anti- α-tubulin mouse monoclonal antibody (1 : 1000; Sigma, St Louis, MO, USA) as a loading control.
The polyvinylidene fluoride membranes were stripped and re-blotted with an anti- β-actin monoclonal antibody (Sigma, St . Louis MO, USA) as a loading control.
Primary antibodies against phosphorylated and total IP3R (1 : 2000, Catalog # 8548 and # 3763, Cell signaling Technology, Danvers, MA, USA) and as a loading control, β-actin (1 : 5000, Catalog # ab6276, Abcam, Cambrige, UK) were used.
The same membranes were analysed with a 1 : 1000 dilution of anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International Inc., Temecula, CA, USA) as a protein loading control.
The same membranes were analysed with a 1 : 2500 dilution of anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal antibody (Chemicon International, Inc., Temecula, CA, USA) as a protein loading control.
β-actin antibody (#3741, CST, USA) was used as a loading control, and anti-caspase-3 9915, CST, USanti-caspase-3 99159502, anti-caspase-3 9915#9272, CST, USA), anti-caspase-975, CST, USA), and anti-PTEN(#9502, CST, USA) (anti-AKT 9272re anti-AKT 9272 CSTl Signaling Technology, Inc (CST).
Western blot analysis was performed to verify efficient knockdown of AKT expression with AKT (Cat number 9272, Cell Signaling, Beverly, MA, USA) and p84 (Cat number GTX70220, Genetex, Irvine, CA, USA) was used as a loading control.
