Journal: Cell Death & Disease

Article Title: NF- κ B inhibition reveals a novel role for HGF during skeletal muscle repair

doi: 10.1038/cddis.2015.66

Figure Lengend Snippet: p65 +/− -CM activates an HGF/Met/GSK3 β pathway in RAW cells. ( a ) Western blot demonstrated that activation of RAW cells in PM induced an increase in pS9-GSK3 β within 30 min (left), a response that was amplified by both WT- (middle) and p65 +/− - CM (right). ( b ) Densitometric analysis revealed that when activated in p65 +/− -CM, the fraction of pS9-GSK3 β increased by 3.5-fold in 30 min, an amount significantly higher than WT-CM and PM groups (* P ⩽0.05). ( c ) Inhibition of Met by SU11274 blocked pS9-GSK3 β in RAW cells 30 min after exposure to LPS and p65 +/− -CM ( d ) in a dose-dependent manner (* versus SF+LPS, + versus no LPS, P <0.05) Data are represented as mean±S.E.M. of at least three independent experiments

Article Snippet: Membranes were incubated with monoclonal antibodies (1 : 1000; Cell Signaling, Danvers, MA, USA) to pS9-GSK3 β , total GSK3 β or polyclonal rabbit anti-HGF (1 : 100; Santa Cruz, Dallas, TX, USA) at 4 °C overnight in 5% milk or BSA in TBST.

Techniques: Western Blot, Activation Assay, Amplification, Inhibition

Journal: Cell Death & Disease

Article Title: NF- κ B inhibition reveals a novel role for HGF during skeletal muscle repair

doi: 10.1038/cddis.2015.66

Figure Lengend Snippet: Hgf upregulation correlates with accelerated muscle regeneration in vivo . ( a ) Hgf expression was significantly upregulated in p65 +/− muscle at 3 days after CTX injury (* P ⩽0.05 versus 3 days WT; + P <0.10 versus day 0 WT). ( b ) Representative images from hemotoxylin and eosin staining indicated that compared with WT muscle, the GAS muscle of p65 +/− mice regenerated more rapidly (arrows) following CTX injury. ( c ) Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis of muscle extracts showed a slight increase in total HGF in uninjured p65 +/− muscles. ( d ) pS9-GSK3 β + macrophages were identified in injured skeletal muscle by immunofluorescent costaining for pS9-GSK3 β (green) and CD68 (red). Arrows indicate examples of colocalization and regions outlined with dashes are digitally enlarged and separated by color channel to the right. ( e ) Quantification of the number of pS9-GSK3 β + macrophages per high power field (HPF, x600) indicated that a significantly higher number of pS9-GSK3 β + /CD68 + macrophages were found in p65 +/ − skeletal muscle compared with WT skeletal muscle at 1, 3 and 5 days after injury ( P ⩽0.05). For ( b ), scale bar: 50 μ m, n =6–8 mice per group. For ( d ), scale bar: 20 μ m, n =3 mice per group. Data are displayed as mean±S.E.M.

Article Snippet: Membranes were incubated with monoclonal antibodies (1 : 1000; Cell Signaling, Danvers, MA, USA) to pS9-GSK3 β , total GSK3 β or polyclonal rabbit anti-HGF (1 : 100; Santa Cruz, Dallas, TX, USA) at 4 °C overnight in 5% milk or BSA in TBST.

Techniques: In Vivo, Expressing, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot