Mouse anti tubulin

The following primary antibodies and dilutions were used for immunofluorescence
studies: mouse anti-Flag (Sigma-Aldrich, F3165) (1:1000), rabbit anti-TOM20 (Santa-Cruz,
sc11415) (1:1000), rabbit anti-PMP70 (Sigma-Aldrich, SAB4200181) (1:1000), mouse
anti-p230 (BD Biosciences, 611281) (1:1000), rabbit anti-Rab7 (Abcam, ab137029)
(1:1000), rat anti-Calnexin (Biolegend, 699401) (1:1000) and mouse anti-Neurofilament H
(Biolegend, 835801). Donkey anti-mouse, Goat anti-mouse IgG1, Goat anti-mouse IgG2a,
Goat anti-rabbit and Goat anti-rat, Alexa Fluor 488, 568 or 647 were used as secondary
antibodies (1:1000) (Invitrogen).
The following primary antibodies and dilutions were used for immunoblot analysis: mouse
anti-Flag (Sigma-Aldrich, F3165) (1:1000), mouse anti-VDAC1 (Abcam, ab14734) (1:1000),
rabbit anti-TMEM63C (Abcam, ab203486) (1:500), rabbit anti-Pex14 (Proteintech,
10594-1-AP) (1:1000), rabbit anti-Calnexin (Proteintech, 10427-1-AP) (1:1000), mouse
anti-Tubulin (Santa-Cruz, sc23948) (1:1000), rabbit anti-VAPB (Atlas, HPA013144) (1:500)
and mouse anti-ACSL4 (Santa-Cruz, sc-365230) (1:1000). Horseradish peroxidase-conjugated
anti-rabbit and anti-mouse IgG (GE Healthcare) were used as secondary antibodies
(1:3000).

Western blot analysis was performed as described previously (Sun et al., 2009 (link)). The C2C12 myotubes or soleus muscles were homogenized in a radio immunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS/1% aprotinin, 50 mM NaF, and 0.1 mM Na₃VO₄) and quantified with Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Equal amounts of total protein per lane were subjected to SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore Corp), which were blocked with 5% non-fat dry milk in Tris-buffer saline (TBS) followed by incubation with primary antibodies: mouse anti-MHC (1:3,000) (R&D Systems, Minneapolis, MN), rabbit anti-MAFbx (1:3,000) (Abcam, Cambridge, UK), rabbit anti-MuRF-1 (1:3,000) and mouse anti-tubulin (1:3,000) (Santa Cruz, Santa Cruz, CA) at 4°C overnight. Then, the membrane was probed with the appropriate horseradish peroxidase-coupled secondary antibody. Immunoactive bands were visualized by enhanced chemiluminescence (Thermo Scientific, Park Ellisville, MO). Densitometry analysis was determined by scanning immunoreactive bands, and intensity values were obtained for further normalization against loading control.