Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: PLoS ONE

Article Title: The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

doi: 10.1371/journal.pone.0067668

Figure Lengend Snippet: A . Ki-67 staining, TUNEL staining, phosphorylated S6 expression, and CD31 staining are demonstrated by immunofluorescence. DAPI was used as a nuclear counterstain. B . Quantification of fluorescence intensity normalized to DAPI. Values atop each bar represent percent intensity relative to the corresponding DAPI image. sEphB4-HSA, soluble EphB4; pS6, phosphorylated S6. C . Phosphorylated Akt and phosphorylated Src expression are demonstrated by immunohistochemistry.

Article Snippet: 5–10 µm sections were fixed in 4% paraformaldehyde, washed in PBS, and incubated in primary anti-Ki-67 (BD Biosciences), anti-CD31 (BD Biosciences), anti-phosphorylated S6 (S235/S236; Cell Signaling, Danvers MA), anti-phosphorylated Akt [S473 (Ref. 34); Cell Signaling], or anti-phosphorylated Src (Y416; Cell Signaling) antibody overnight at 4°C.

Techniques: Staining, TUNEL Assay, Expressing, Immunofluorescence, Fluorescence, Immunohistochemistry

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: Depletion of CLCa, but not CLCb, specifically inhibits cell spreading and motility. (A) HeLa cells were transiently transfected with Qiagen CLCa (KD-a), CLCb (KD-b) or negative control (NC) siRNAs (see Materials and Methods for details) and analyzed for spreading on collagen IV at indicated time points. Cellular spreading was denoted as fold change over spreading of NC-transfected cells at 30 min. Data from 5–7 plating experiments are summarized together. (B) HeLa cells were transiently transfected with Dharmacon CLCa siRNA with or without siRNA-resistant CLCa DNA (CLCaR) and analyzed for spreading as described for A. (C,D) H1299 and HEK293 cells were transiently transfected with Dharmacon CLCa, Qiagen CLCb or NC siRNAs and analyzed for spreading as described in A. (E) siRNA-transfected H1299 cells were plated on collagen IV-coated dishes and grown to confluence. After scratching three of four wounds per siRNA, cells were allowed to recover for 20 h and analyzed for wound closure. Complete wound healing was set as 100%. The graph represents data from three independent experiments. (F) Velocity and directionality of siRNA-transfected HEK293 cells were analyzed by time-lapse microscopy and ImageJ chemotaxis software. 20–50 cells per siRNA were analyzed in two independent experiments. Western blots are presented as confirmation of knockdown. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Negative Control, Time-lapse Microscopy, Chemotaxis Assay, Software, Western Blot

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCb-deficient clathrin structures in protrusions of spreading cells. HeLa cells were plated for 60 min, then fixed and processed as indicated. (A,B) Cells immunostained for CHC (ab21679) and CLCb and imaged by confocal microscopy at the adherent surface (A) or by TIRF microscopy (B). Peripheral CHC structures comparatively deficient in CLCb signal are detectable in the confocal image (insets), and are more apparent by TIRF imaging (B) as multiple CHC-containing puncta (short arrowheads) and regions with less punctate staining (long arrowheads). Scale bar: 10 µm (main image in A); 2 µm (magnified images in A and B). Results are representative of five experiments. (C) Both F-actin (stained with SiR-actin) and cortactin are present in protrusions containing small CHC puncta, as imaged by confocal microscopy. Scale bars: 5 µm (main image); 2 µm (magnifications). (D) TIRF microscopy reveals F-actin (Alexa Fluor 546–phalloidin) and CHC staining in a protrusion (arrowhead). Scale bar: 2 µm. (E) Spreading region in the periphery of a KD-a cell expressing HA–CLCa-R and immunostained for HA and CLCb. TIRF microscopy reveals diffuse (arrows) and punctate structures (arrowheads) containing CLCa with low or undetectable levels of CLCb. Scale bar: 2 µm. Representative of five independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Confocal Microscopy, Microscopy, Imaging, Staining, Expressing