Table 1.
Name and sequences of primers.
Table 2.
Maximal citrate synthase (CS) activity in μmol.min−1.g−1 wet wt.
Fig 1.
ATGL, CGI-58 and G0S2 mRNA expression in the three skeletal muscles (SOL, RG, and WG and heart, n = 9).
Data were calculated according to the delta delta CT method relative to WG as explained in Materials and Methods. Data are reported as mean±SE, and there were no statistically significant differences within the skeletal muscles (p<0.05). SOL, soleus; RG, red gastrocnemius; WG, white gastrocnemius.
Fig 2.
A. ATGL (n = 8) and CGI-58 (n = 7) protein content in the three skeletal muscles (SOL, RG, and WG and heart).
Ponceau stain for total protein was used as a loading control. Data are reported as mean±SE and bars with the same letter are not significantly different within the skeletal muscles (p<0.05). Insets: representative blots: lane 1, WG (white gastrocnemius); lane 2, RG (red gastrocnemius); lane 3, SOL (soleus). B. ATGL and CGI-58 western blot protein bands compared to representative Ponceau bands as equal loading control.
Fig 3.
Establishing that G0S2 recognized proteins that migrated at both 15 kDa and 25 kDa.
As described in Methods, blots were detected with two antibodies directed against different regions of G0S2 (Santa Cruz, sc-133423, sc-133424) but for the ‘Blocked’ panel, but specific blotting peptides were used (Santa Cruz, sc-133423 P. sc-133424 P). Images were captured at the same time to eliminate bias placed upon exposure times and image editing. Three lanes on the left were mouse SOL (n = 3), three lanes on the right were rat SOL (n = 3). Notice both the 25kDa and the 15kDa band show in both species, and both bands disappear with the antibody pre-incubation with specific blocking peptides.
Fig 4.
A. G0S2 Overexpressing lysate compared to SOL at 25kDa.
Whole blot present shows that the 25kDa band is prominent in SOL with the ~13kDa band present in the lysate and no 25kDa band present. B. G0S2 Overexpressing lysate compared to SOL at 15kDa. Notice that the 15kDa band is now visible in SOL lane with the 25kDa cut out prior to exposure. C. Comparing a wild type to global G0S2 knockout mouse mixed hindlimb muscle for the G0S2 protein (n = 2). Notice that in both the WT (wild type) and the KO (knockout) lanes that the 25kDa band is present, yet the 15kDa band is only present in the WT. The top panel represents the 25kDa band, with the bottom panel representing the 15kDa band from the same blot.
Fig 5.
A. G0S2 protein content in the three skeletal muscles (SOL, RG, and WG and heart, n = 8).
Ponceau stain was used as a loading control. Data are reported as mean±SE and bars with the same letter are not significantly different within the skeletal muscles (p<0.05). Insets: representative blots: lane 1, WG (white gastrocnemius); lane 2, RG (red gastrocnemius); lane 3, SOL (soleus). B. 15kDa G0S2 western blot protein bands compared to representative Ponceau bands as equal loading control.
Fig 6.
Calculated G0S2-to-ATGL (n = 8) and CGI-58-to-ATGL (n = 7) ratios.
Data are reported as mean±SE and bars with the same letter are not significantly different between the skeletal muscles (p<0.05). Please note that ratios were calculated from raw integrations in arbitrary units. Blots were always run in same rat pairs for all the proteins and tissues and every effort to keep exposure times etc., as consistent as possible to keep variation between blots to a minimum. SOL, soleus; RG, red gastrocnemius; WG, white gastrocnemius.