Proteinase and phosphatase inhibitors

Protein isolation of murine heart tissue was performed with the TissueLyser LT (Qiagen) using RIPA buffer with proteinase and phosphatase inhibitors (Sigma-Aldrich). Samples were incubated at 4°C for 30 min followed by sonification. Protein concentrations were measured via BCA assay (Thermo Fisher Scientific) according to manufacturer’s protocol.
100 µg total protein was separated on a 12% SDS-gel and transferred on nitrocellulose membrane in transfer buffer containing 18% methanol for 1 h. Membranes were blocked in 5% milk in TBS containing 0.05% Tween-20 for 1 h, afterwards primary antibodies were incubated over-night at 4°C (FGF23-6310 Goat mAb, dilution 1:500, Immutopics; GAPDH 14C10 Rabbit mAb, dilution 1:1,000, Cell Signaling Technology). After washing, secondary antibodies were incubated for 1 h at room temperature (Goat anti-rabbit IgG HRP, dilution 1:1,000, Santa Cruz; Donkey anti-goat IgG HRP, dilution 1:2000, R and D Systems). ECL development was done with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and signals were detected with the Odyssey FC Imaging System (LI-COR Bioscience). Expression levels were quantified with the Image Studio Lite 5.2 software (LI-COR Bioscience).

The treated cells were washed with cold PBS, resuspended with RIPA buffer containing proteinase and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO), and lysed at 4 °C for 1 h. The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA). Equivalent amounts of total protein were separated on 12% gels by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% defatted milk for 1 h and then immunoblotted with primary antibody anti-ATG5 (#12994, Cell Signaling Technology, Danvers, MA), anti-Beclin1 (#37385, Cell Signaling Technology, Danvers, MA), anti-Bax (#2774, Cell Signaling Technology, Danvers, MA), anti-caspase-3 (#9662, Cell Signaling Technology, Danvers, MA) or anti-GAPDH (#1310016, Thermo Fisher Scientific, Waltham, MA) overnight at 4 °C. The membranes were washed and then incubated with specific secondary antibodies (#2999, Cell Signaling Technology, Danvers, MA). Finally, the blots were visualized by chemiluminescence (ECL; Forevergen Biosciences Center, Guangzhou, China).

Transfected and non-transfected HEK293 and NBL cell cultures were lysed in RIPA buffer complementing with appropriate concentrations (1μM) of proteinase and phosphatase inhibitors (Sigma). Cell lysates containing proteins were separated on 4–15% or 10% SDS polyacrylamide gels and transferred to a PVDF membrane (Biorad) using transfer buffer containing 20% methanol in 25mM Tris-HCl and 192mM glycine. For transfer of large proteins such as pITPR1 western blot, 5–6% Tris-HCl/SDS polyacrylamide gel and 10% methanol in transfer buffer were used to increase the transfer efficiency. Blocking of the membrane was done with 5% skimmed milk in Tris-buffered saline (TBS) with 0.1% tween-20 for 1 h at room temperature followed by wash with TBS containing tween-20 and incubation with primary antibodies at 4°C. The blots were then incubated for 1 h at HRP-conjugated secondary antibodies at room temperature (Santa Cruz laboratories). Pierce Super Signal substrate (Thermofisher Scientific, Rockford, IL), was used to visualize bands. Primary antibodies used were as follows: anti-CA8 (Santa Cruz, Santa Cruz, CA), anti-V5 (Invitrogen), anti-pITPR1 (Cell Signalling Tech.) and anti-ITPR1 (Cell Signaling Technology, Ser-1755), anti-FLAG (Sigma Aldrich), anti-FLAG (Aves), anti-Vinculin (Abcam) and anti-β-actin (Cell Signaling Tech.).