Srx 101a film processor

EGFR was immunoprecipitated with 108 EGFR antibody bound to protein G Sepharose Fast Flow beads (Sigma). Immunoprecipitates were extensively washed and analysed by western blotting.
For western blotting, cells were lysed in lysis buffer [20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40 (or Triton-X100), pH 7.4 plus protease inhibitor cocktail (Calbiochem set I) and phosphatase inhibitor cocktail (Calbiochem set II)]; lysates were fractioned by SDS–PAGE on 10% gels under reducing conditions and immunoblotted onto nitrocellulose membranes. Bands were detected by using enhanced chemiluminescence (Pierce) and exposed films developed on a SRX-101A Film Processor (Konica).
See Supplementary Figure 6 for uncropped scans of blots.

Flash-frozen hearts were pulverized using a tissue crusher and protein was isolated in RIPA buffer (MilliporeSigma, R0278) containing protease and phosphatase inhibitors (Roche, 04693159001 and 04906837001). To break genomic DNA, samples were sonicated using Bioruptor Pico (Diagenode) for 10 cycles of 30-second sonication on and 30-second sonication off. Subsequently, samples were centrifuged at 12,000g for 20 minutes at 4°C to pellet cell debris. Protein concentration was determined by BCA assay (Thermo Fisher, 23225), and equal amounts of protein among samples were used for regular Western blot and transferred to polyvinylidene fluoride membrane (Millipore, IPVH00010).
Blocking and antibody incubation were performed in 5% milk or 5% BSA in TBS-Tween 0.1%. The following primary antibodies were used: LEMD2 (MilliporeSigma, HPA017340, 1:500) and GAPDH (MilliporeSigma, MAB374, clone 6C5; 1:500). Horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies were used (Bio-Rad, 1706515 and 1706516, 1:5000). Immunodetection was performed using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., sc2048) using a SRX-101A film processor (Konica Minolta) and ChemiDoc MP Imaging system (Bio-Rad). See complete unedited blots in the supplemental material.

Cells were lysed in 1X RIPA buffer with protease and phosphatase inhibitors. Fifteen micrograms of protein was separated on 4–10% acrylamide/bisacrylamide gels, transferred onto PVDF membranes, and blocked for 1 hr in 5% milk in 1× TBST. The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature. Following primary antibody incubation, the membranes were washed three times with 1× TBST buffer at room temperature and probed with rabbit or mouse horseradish peroxidase-linked secondary antibody (1:5000; ECL NA931 mouse, NA934V Rabbit). The western blot signal was detected using the ECL Prime (RPN322) kit and the blot was exposed to an X-Ray film, which was developed using the Konica Minolta SRX 101A film processor.

To estimate total EV protein, EVs were suspended in RIPA buffer (200 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA with protease and phosphatase inhibitors) and lysed on ice for 10 min. Protein estimation was performed with the Pierce BCA protein assay kit against a BSA standard curve. Lysed EVs were analyzed in triplicate and the values were averaged for the total protein estimate. For EV protein analysis, 25 μg of total EV protein was lysed as mentioned above. Protein was denatured in SDS loading buffer at 95°C for 10 m. Samples were separated on 4–15% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with 5% milk in TBS-T (1X TBS with 0.1% Tween-20) for 1 h at RT, then probed with primary antibody at 1:1,000 with overnight incubation at 4°C with gentle rocking. Membranes were washed three times with TBS-T for 10 m each. Secondary antibody was added at 1:10,000 and incubated at 4°C for 2 h with gentle rocking. Membranes were washed three times with TBS-T and incubated with ECL^TM Western blotting detection reagents (GE Healthcare, United Kingdom) for 1 m at RT. Membranes were exposed to film and developed with a SRX-101A film processor (Konica Minolta, Japan).