Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 1. Phosphorylation of (A) MLC peptide and (B) SAMS peptide by the AMPK and smMLCK preparations in vitro. Initial rates of 32P-incorporation by 0.07 lg of rat liver AMPK (4), 0.07 lg of recombinant AMPK (h) or 1 lg of smMLCK (s) into the MLC peptide were 137 ± 18, 105 ± 7 and 194 ± 12 lUnits/50 ll of reaction mixture, respectively, and into the SAMS peptide were 215 ± 9, 201 ± 26 and 2.3 ± 1.1 lUnits/50 ll of reaction mixture, respectively.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, In Vitro, Recombinant

Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 2. Phosphorylation of (A) GST-ACC2 peptide and (B) MLC by the AMPK and smMLCK preparations in vitro. Initial rates of 32P-incorporation by 0.07 lg of rat liver AMPK (4), 0.07 lg of recombinant AMPK (h) or 1 lg of smMLCK (s) into GST- ACC2 were 10.5 ± 1.3, 5.7 ± 0.9 and 0.3 ± 0.1 lUnits/50 lL of reaction mixture, respectively, and into MLC were 12.0 ± 0.9, 1.3 ± 0.4 and >109 ± 4 lUnits/50 ll of reaction mixture, respectively.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, In Vitro, Recombinant

Journal: FEBS letters

Article Title: Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes.

doi: 10.1016/j.febslet.2008.11.022

Figure Lengend Snippet: Fig. 3. Effect of H1152 on A769662-induced AMPK Thr172, ACC Ser221 and MLC Ser19 phosphorylation in MDCK cells. MDCK cells were incubated in the presence or absence of 20 lM A769662 for 30 min with or without 60 min of pre-treatment with 1 lM H1152 Rho kinase inhibitor. Cell lysates were analyzed by immunoblotting with anti- phospho Thr172 AMPK a-subunit, anti-phospho Ser221 ACC or anti-phospho Ser19 MLC antibodies along with the appropriate anti-full length protein antibodies as loading controls for detection by chemiluminescence and scanning densitometry. *Indicates a significant difference (P < 0.05, unpaired t-test) with respect to the value corresponding to treatment without inhibitor.

Article Snippet: CaM, antifull-length smMLCK antibody (clone K36, Sigma), GST-human acetyl-CoA carboxylase (ACC)2, purified rat liver AMPK (Upstate), anti-rabbit Immunoglobulin G conjugated to IR dye 800 (Rockland Inc., Philadelphia, PA), anti-sheep Immunoglobulin G conjugated to Alexa 680 (Molecular Probes), anti-phospho-Thr172 AMPK a-subunit and anti-phospho Ser19 MLC antibodies (Bioké), anti-phospho Ser221 ACC2, anti-full-length ACC2 antibodies (Upstate), anti-fulllength MLC antibody (Sigma) and enhanced chemiluminescence reagents (Thermofisher) were from the indicated sources.

Techniques: Phospho-proteomics, Incubation, Western Blot