Mouse anti smn

Fibroblast cultures were fixed in 4% paraformaldehyde for 20 minutes and then incubated with 3% of triton (10 min) and 5% BSA (45 min) solutions. Primary antibody mouse anti-SMN (BD Transduction Laboratories) at 1 : 100 was incubated O/N at 4°C and secondary antibody Alexa Fluor 546 goat anti-mouse together with Alexa Fluor 488-conjugated phalloidin (both at 1 : 200; Invitrogen) was incubated for 1 h at room temperature. Images were taken with a confocal microscopy, Radiance 2100 microscope (Biorad Laboratories, Hercules, CA).

Protein lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer and brief sonication. A total of 30 μg of homogenized protein samples was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane in iBlot™ gel transfer stack using the iBlot dry blotting system (Invitrogen), as described previously [33 (link)].
Membranes were blocked with 5% nonfat dry milk and incubated overnight with primary antibodies. Mouse anti-β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-SMN (1:5000; BD Transduction Laboratories, Franklin Lakes, NJ, USA) primary antibodies were used to detect β-actin and SMN1 proteins, respectively. β-actin was used as an endogenous reference protein and as the relative denominator for Western blotting. Anti-mouse IgG (Cell Signaling #7076 Anti-mouse IgG, HRP-linked) was used as a secondary antibody.
Chemiluminescent signals produced using Immobilon™ Western chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA) were detected using the Bio-Rad ChemiDoc Touch MP imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The signal intensity of the membrane was determined using ImageJ ver. 2.0. Western blotting experiments and signal-intensity analyses were performed in triplicate.