Figure 5.
In muscle cells, paxillin is present at the NMJ and promotes the regulation of MT acetylation.(A and C) Isolated fibers (A) and cross sections (C) of TA muscles from 2-mo-old mice were double-stained with an antibody against paxillin (in red) and with α-BTX–A488 (in green). (B and D) The fluorescence intensity of each staining was plotted as a function of the distance (based on the blue line scans in A and C, respectively). The green curve corresponds to α−BTX–A488 staining and the red curve to paxillin staining. (E) Western blot showing the co-immunoprecipitation of endogenous HDAC6 and paxillin in TA muscle cells. (F and G) Representative images (F) and quantitation (G) of a PLA performed in isolated fibers of TA muscle with protein-specific antibody pairs as indicated. Cells were counterstained with α-BTX–A488 in green (n = number of synaptic domains quantified). PLA-positive spots are shown in red. The arrowhead shows the colocalization of α-BTX and PLA. Means ± SEM. ***, P < 0.001; Mann-Whitney U test. (H) Schematic representation of the experimental time course. (I–L) Myoblasts were transfected with either shRNA-Control (pLKO), shRNA against paxillin (shPXN), GFP alone, or PXN-GFP for 24 h. (I and K) Representative Western blots showing endogenous paxillin, acetylated tubulin (ac-tubulin), GFP, and β-tubulin expression. GAPDH was used as a loading control. (J and L) Quantification of acetylated tubulin protein level, normalized to β-tubulin. Paxillin protein level quantification was standardized to GAPDH. Means ± SEM (n = number of Western blots quantified). The baseline was established to 1 for the control condition for each individual Western blot. Dashed lines indicate edges of cells. Scale bars: 25 µm (A, C, and F); inset magnifications (C and F; boxes 1, 2, and 3) 5 µm. Fluo, fluorescence; β-tub, β-tubulin; IP, immunoprecipitation; Mr(K), relative molecular weight in kiloDalton; ac-tub and ac-tubulin, acetylated tubulin.

In muscle cells, paxillin is present at the NMJ and promotes the regulation of MT acetylation. (A and C) Isolated fibers (A) and cross sections (C) of TA muscles from 2-mo-old mice were double-stained with an antibody against paxillin (in red) and with α-BTX–A488 (in green). (B and D) The fluorescence intensity of each staining was plotted as a function of the distance (based on the blue line scans in A and C, respectively). The green curve corresponds to α−BTX–A488 staining and the red curve to paxillin staining. (E) Western blot showing the co-immunoprecipitation of endogenous HDAC6 and paxillin in TA muscle cells. (F and G) Representative images (F) and quantitation (G) of a PLA performed in isolated fibers of TA muscle with protein-specific antibody pairs as indicated. Cells were counterstained with α-BTX–A488 in green (n = number of synaptic domains quantified). PLA-positive spots are shown in red. The arrowhead shows the colocalization of α-BTX and PLA. Means ± SEM. ***, P < 0.001; Mann-Whitney U test. (H) Schematic representation of the experimental time course. (I–L) Myoblasts were transfected with either shRNA-Control (pLKO), shRNA against paxillin (shPXN), GFP alone, or PXN-GFP for 24 h. (I and K) Representative Western blots showing endogenous paxillin, acetylated tubulin (ac-tubulin), GFP, and β-tubulin expression. GAPDH was used as a loading control. (J and L) Quantification of acetylated tubulin protein level, normalized to β-tubulin. Paxillin protein level quantification was standardized to GAPDH. Means ± SEM (n = number of Western blots quantified). The baseline was established to 1 for the control condition for each individual Western blot. Dashed lines indicate edges of cells. Scale bars: 25 µm (A, C, and F); inset magnifications (C and F; boxes 1, 2, and 3) 5 µm. Fluo, fluorescence; β-tub, β-tubulin; IP, immunoprecipitation; Mr(K), relative molecular weight in kiloDalton; ac-tub and ac-tubulin, acetylated tubulin.

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