Anti tubulin

Tissue samples of 20–25 mg were taken, and 500μL of RIPA lysis buffer containing 1 mM of PMSF and 0.02% protease phosphatase inhibitors were added for homogenization. Subsequently, the homogeneous liquid was incubated at 4 °C for 30 min. Insoluble substances were removed from the suspension by 12,000× g of centrifugation for 15 min, and total protein concentration was quantified by BCA protein assay (Beyotime, Shanghai, China). The protein suspension was electrophoresed in 12% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) by the Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA, USA), then blocked with 5% non-fat dry milk in Tris-buffered saline for 2 h at room temperature. Finally, the anti-TMEM8c (dilution 1:500; NOVUS, Littleton, CO, USA) and anti-Tubulin (Abcam, Cambridge, MA, USA) were added separately and incubated overnight at 4 °C (or 2 h at room temperature), followed by 1 h with Alexa Fluor^® 488 secondary antibody (Abcam, Cambridge, MA, USA). After incubating the membrane with chemiluminescence reagent (Beyotime, Shanghai, China), the protein expression was detected by a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA), and anti-Tubulin (Abcam, Cambridge, MA, USA) was used as a housekeeping protein for normalizing Myomaker.

Cell pellets were lysed in RIPA buffer (Pierce) supplemented with protease inhibitors (Roche) and incubated for 20 min at 4°C. After centrifugation at 21,000 g for 10 min at 4°C, protein concentration was quantified using a Direct Detect Infrared Spectrometer (Merck). Ten to 20 µg total protein was separated on a NuPAGE 4–12% Bis-Tris denaturing gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using an iBLot2 dry transfer (Invitrogen). Primary antibody incubations were performed overnight at 4°C and IRDye 680RD- and 800CW-conjugated secondary antibodies (LI-COR) were incubated for 45 min at room temperature. Primary antibodies used are as follows: anti-FLAG (1:2500; Sigma F1804), anti-HA (1:2500; Cell Signaling Technology C29F4), anti-Tubulin (1:2500; abcam ab18251), anti-Tubulin (1:2500; abcam ab44928), anti-Lamin (1:200; Developmental Studies Hybridoma Bank ADL67.10), anti-His3 (1:1000; abcam ab10799), anti-DYNLL1 (1:1000; abcam ab51603), anti-Piwi (1:2500) (Brennecke et al., 2007 (link)), and anti-Panx antibody (1:20) (Sienski et al., 2015 (link)). Images were acquired using an Odyssey CLx scanner (LI-COR) and processed/quantified in Image Studio Lite (LI-COR).

Fourty adult animals were transferred to an Eppendorf tube containing 20 μl of water. 20 μl of 2xSDS buffer were added, and the sample was incubated for 5 min at 95°C. Genomic DNA was digested by adding 1 μl of DNase (Qiagen) and incubating for 5 min at room temperature, followed by a 5 min inactivation at 95°C. Proteins were separated by SDS PAGE on 4–12% acrylamide gradient gels and blotted onto PVDF membranes. After blocking non-specific binding sites with 5% milk or bovine serum albumin in TBST (20 mM Tris, 150mM NaCl, 0.1% Tween 20), the membranes were incubated with the primary antibody diluted in TBST containing 5% milk overnight at 4°C. After incubation with HRP-conjugated secondary antibodies, the protein bands were viualized by chemiluminescence using the SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Scintific). Quantification was performed by measuring the band intensities using Fiji’s measurement tools [56 (link)]. Band intensities were first normalized to the alpha-tubulin loading controls and then to the maximum value in each experiment. The following antibodies were used: anti-SUMO-1 1:500 (S5446 Sigma), anti-Flag 1:3000 (Sigma F3165-1MG), anti-Tubulin 1:10 000 (Abcam ab18251), HRPGoat anti-Rabbit 1:2000 (Jackson ImmunoReserach 111-035-144) and HRP Goat anti-Mouse 1:2000 (Jackson ImmunoReserach 115-035-146).