Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Sequencing

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: Reagents used for in vitro experiments.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Stem Cells International

Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

doi: 10.1155/2019/1513526

Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: ER-mediated actin cytoskeletal remodeling induces suppressive cortical actin bundles (SCABs). a Leading edge kymography in representative time-lapse movies (Supplementary Movie ) of control and E2-treated MCF7 cells (cultured under hormone starvation conditions), and control and ER-inhibited cells (cultured in regular media). Left panels indicate position at which kymographs were registered (line), and middle panels show minimum intensity projections from entire time series (Min. Proj.); scale bar is 10 µm. Right panels show corresponding kymographs; vertical scale bar is 10 µm; horizontal scale bar is 5 min. b Membrane ruffle quantification; mean ± s.d; * p = 0.03, *** p = 0.0005, **** p < 0.0001 (Welch’s t test). c Ruffling speed quantification; mean ± s.e.m; * p = 0.01, *** p = 0.0007, **** p < 0.0001 (Welch’s t test). Data from three independent experiments ( n ≥ 45 cells per group). d Leading edge kymography in representative time-lapse movies (Supplementary Movie ) of T47D cells. e Membrane ruffle quantification; mean ± s.d; * p = 0.03, *** p = 0.0002, **** p < 0.0001 (Welch’s t test). f Ruffling speed quantification; mean ± s.e.m; * p = 0.01, ** p = 0.004, **** p < 0.0001 (Welch’s t test). Data from two independent experiments ( n ≥ 30 cells per group). g Volumetric analysis of leading edge actin. Illustrative confocal z-series and corresponding 3D binary mask, demarcated as the area within 3 µm of cell edge and used to quantify leading edge fluorescence intensity. h Maximum intensity projections of z-series of E2-treated or fulv-treated MCF7 cells (Supplementary Movie ), and 3D reconstructions of boxed areas; F-actin (red), Arp2/3 (green), and pMLC (magenta). Scale bar is 10 µm. i Quantification of leading edge Arp2/3 and pMLC in MCF7 cells. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups ( p < 0.001). Arp2/3 values are not significantly different between groups. j Maximum intensity projections of z-series of E2-treated or tam-treated T47D cells and 3D reconstructions of boxed areas. F-actin is red, Arp2/3 in green, and pMLC in magenta. Scale bar is 10 µm. k Quantification of leading edge Arp2/3 and pMLC in T47D cells. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups ( p < 0.001). Arp2/3 values are not significantly different between groups

Article Snippet: The following antibodies were used: human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling), hHuman anti-Arp2/3 clone no.13C9 (Millipore), human anti-pan cytokeratin no. AE1/AE3 (Dako), human anti-ER no. H4624 (Invitrogen), human anti-EVL no. HPA018849 (Sigma Prestige), human anti-EVL (a gift from Frank Gertler), and human anti-Actin (Abcam, ab3280).

Techniques: Cell Culture, Fluorescence

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: SCABs suppress leading edge motility in a myosin contractility-dependent manner. a Leading edge kymography in MCF7 cells (top row) and T47D cells (bottom row) expressing iRFP-Lifeact (black) and MLC-mRuby2 (magenta) before and after dissolving SCABs using 25 µM ROCK inhibitor. Left panels are images from TIRF microscopy time-lapse series before and after the addition of ROCK inhibitor (Supplementary Movie ). Scale bar is 10 µm. Line shows the leading edge position at which kymographs were registered. Right panel shows MLC kymograph (denoting SCABs) superimposed over Lifeact kymograph (demarcating the leading edge). Vertical scale bar is 10 µm and horizontal scale bar is 10 min. b Immunofluorescence of SCAB in MDCK epithelial cells and Caco-2 human intestinal epithelial cells. Representative images for F-actin, pMLC, and Arp2/3. Scale bar is 10 µm. c Leading edge kymography in MDCK cells (top row) and Caco-2 (bottom row) cells expressing iRFP-Lifeact (black) and MLC-mRuby2 (magenta) before and after dissolving SCABs using 25 µM ROCK inhibitor. Scale bar is 10 µm. Line shows the leading edge location at which the kymograph was registered. Right panel shows MLC kymograph (denoting SCABs) superimposed over Lifeact kymograph (demarcating the leading edge). Vertical scale bar is 10 µm and horizontal scale bar is 10 min

Article Snippet: The following antibodies were used: human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling), hHuman anti-Arp2/3 clone no.13C9 (Millipore), human anti-pan cytokeratin no. AE1/AE3 (Dako), human anti-ER no. H4624 (Invitrogen), human anti-EVL no. HPA018849 (Sigma Prestige), human anti-EVL (a gift from Frank Gertler), and human anti-Actin (Abcam, ab3280).

Techniques: Expressing, Microscopy, Immunofluorescence

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: EVL promotes ER-mediated actin remodeling. a Maximum intensity projections of z-series of control and E2-treated LKO and EVL KD MCF7 cells. Scale bar is 10 µm. b Quantification of leading edge Arp2/3 and pMLC. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m.). Three-way ANOVA shows significant differences between LKO and EVL KD groups at p = 0.08 (df = 1). Two-way ANOVA shows significant difference in pMLC levels at p < 0.05 between control and E2-treated cells in the LKO group, but not in the EVL KD group. No significant differences in Arp2/3 levels were observed at p < 0.05. ** p < 0.01. c Maximum intensity projections of z-series of control and fulv-treated eGFP and eGFP-EVL MCF7 cells. Scale bar is 10 µm. Scale bar is 5 µm for inset showing eGFP-EVL and pMLC. Larger fields of view from the same group are given in Supplementary Fig. . d Quantification of leading edge Arp2/3 and pMLC. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m.). Three-way ANOVA shows significant differences between eGFP and eGFP-EVL groups at p < 0.0001 (df = 1). Two-way ANOVA shows significant difference in pMLC levels at p < 0.05 between control and fulv-treated cells in the eGFP group, but not in the eGFP-EVL group. No significant differences in Arp2/3 levels were observed at p < 0.05. * p < 0.05. e Analysis of EVL localization at SCABs by iPALM in MCF7 cells expressing mEos2-EVL. Left panels show single and merged images of F-actin staining and mEos2-EVL; boxes indicate ROIs. Arrows indicate en face orientation of orthogonal projections. Orthogonal projections of ROIs are shown with corresponding histogram highlighting z-localization of F-actin (red) and mEos2-EVL (cyan); horizontal axes represent molecular counts (mol. cts.) for mEos2-EVL (top) and F-actin (bottom). Merge image scale bar is 5 µm. Orthogonal projection scale bar is 150 nm. f Leading edge kymography in control and iRFP670-EVL (green) expressing MCF7 cells (top and bottom rows, respectively), with eGFP-Lifeact (black) and MLC-mRuby2 (magenta), before and after ROCK inhibitor (50 µM) treatment (Supplementary Movie ). Left panels are images from time-lapse series before and after treatment. Scale bar is 10 µm. Line shows leading edge location at which kymographs were registered. Inset shows EVL channel separately. Right panel shows kymograph. Vertical scale bar is 10 µm. Horizontal scale bar is 10 min. n.s. nonsignificant

Article Snippet: The following antibodies were used: human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling), hHuman anti-Arp2/3 clone no.13C9 (Millipore), human anti-pan cytokeratin no. AE1/AE3 (Dako), human anti-ER no. H4624 (Invitrogen), human anti-EVL no. HPA018849 (Sigma Prestige), human anti-EVL (a gift from Frank Gertler), and human anti-Actin (Abcam, ab3280).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: ER-mediated actin cytoskeletal remodeling induces suppressive cortical actin bundles (SCABs). a Leading edge kymography in representative time-lapse movies (Supplementary Movie ) of control and E2-treated MCF7 cells (cultured under hormone starvation conditions), and control and ER-inhibited cells (cultured in regular media). Left panels indicate position at which kymographs were registered (line), and middle panels show minimum intensity projections from entire time series (Min. Proj.); scale bar is 10 µm. Right panels show corresponding kymographs; vertical scale bar is 10 µm; horizontal scale bar is 5 min. b Membrane ruffle quantification; mean ± s.d; * p = 0.03, *** p = 0.0005, **** p < 0.0001 (Welch’s t test). c Ruffling speed quantification; mean ± s.e.m; * p = 0.01, *** p = 0.0007, **** p < 0.0001 (Welch’s t test). Data from three independent experiments ( n ≥ 45 cells per group). d Leading edge kymography in representative time-lapse movies (Supplementary Movie ) of T47D cells. e Membrane ruffle quantification; mean ± s.d; * p = 0.03, *** p = 0.0002, **** p < 0.0001 (Welch’s t test). f Ruffling speed quantification; mean ± s.e.m; * p = 0.01, ** p = 0.004, **** p < 0.0001 (Welch’s t test). Data from two independent experiments ( n ≥ 30 cells per group). g Volumetric analysis of leading edge actin. Illustrative confocal z-series and corresponding 3D binary mask, demarcated as the area within 3 µm of cell edge and used to quantify leading edge fluorescence intensity. h Maximum intensity projections of z-series of E2-treated or fulv-treated MCF7 cells (Supplementary Movie ), and 3D reconstructions of boxed areas; F-actin (red), Arp2/3 (green), and pMLC (magenta). Scale bar is 10 µm. i Quantification of leading edge Arp2/3 and pMLC in MCF7 cells. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups ( p < 0.001). Arp2/3 values are not significantly different between groups. j Maximum intensity projections of z-series of E2-treated or tam-treated T47D cells and 3D reconstructions of boxed areas. F-actin is red, Arp2/3 in green, and pMLC in magenta. Scale bar is 10 µm. k Quantification of leading edge Arp2/3 and pMLC in T47D cells. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m). By two-way ANOVA, interaction analysis shows significant differences at p < 0.0001 (df = 3); pMLC values are significantly different in E2-treated group compared to other groups ( p < 0.001). Arp2/3 values are not significantly different between groups

Article Snippet: The actin cytoskeleton was stained with Alexa Fluor-conjugated phalloidin (Alexa Fluor 568-Phalloidin or Alexa Fluor 647-Phalloidin; Thermo Fisher) diluted to 2.5% in phosphate-buffered saline (PBS); nuclei were stained with Hoechst 33342 (Thermo Scientific); pMLC was immunolabeled using human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling) at 1:100 dilution; Arp2/3 was immunolabeled using human anti-Arp2/3 clone no.13C9 (Millipore) at 1:200 dilution; EVL was immunolabeled using human anti-EVL (a gift from Frank Gertler) at 1:50 dilution.

Techniques: Cell Culture, Fluorescence

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: SCABs suppress leading edge motility in a myosin contractility-dependent manner. a Leading edge kymography in MCF7 cells (top row) and T47D cells (bottom row) expressing iRFP-Lifeact (black) and MLC-mRuby2 (magenta) before and after dissolving SCABs using 25 µM ROCK inhibitor. Left panels are images from TIRF microscopy time-lapse series before and after the addition of ROCK inhibitor (Supplementary Movie ). Scale bar is 10 µm. Line shows the leading edge position at which kymographs were registered. Right panel shows MLC kymograph (denoting SCABs) superimposed over Lifeact kymograph (demarcating the leading edge). Vertical scale bar is 10 µm and horizontal scale bar is 10 min. b Immunofluorescence of SCAB in MDCK epithelial cells and Caco-2 human intestinal epithelial cells. Representative images for F-actin, pMLC, and Arp2/3. Scale bar is 10 µm. c Leading edge kymography in MDCK cells (top row) and Caco-2 (bottom row) cells expressing iRFP-Lifeact (black) and MLC-mRuby2 (magenta) before and after dissolving SCABs using 25 µM ROCK inhibitor. Scale bar is 10 µm. Line shows the leading edge location at which the kymograph was registered. Right panel shows MLC kymograph (denoting SCABs) superimposed over Lifeact kymograph (demarcating the leading edge). Vertical scale bar is 10 µm and horizontal scale bar is 10 min

Article Snippet: The actin cytoskeleton was stained with Alexa Fluor-conjugated phalloidin (Alexa Fluor 568-Phalloidin or Alexa Fluor 647-Phalloidin; Thermo Fisher) diluted to 2.5% in phosphate-buffered saline (PBS); nuclei were stained with Hoechst 33342 (Thermo Scientific); pMLC was immunolabeled using human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling) at 1:100 dilution; Arp2/3 was immunolabeled using human anti-Arp2/3 clone no.13C9 (Millipore) at 1:200 dilution; EVL was immunolabeled using human anti-EVL (a gift from Frank Gertler) at 1:50 dilution.

Techniques: Expressing, Microscopy, Immunofluorescence

Journal: Nature Communications

Article Title: The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

doi: 10.1038/s41467-018-05367-2

Figure Lengend Snippet: EVL promotes ER-mediated actin remodeling. a Maximum intensity projections of z-series of control and E2-treated LKO and EVL KD MCF7 cells. Scale bar is 10 µm. b Quantification of leading edge Arp2/3 and pMLC. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m.). Three-way ANOVA shows significant differences between LKO and EVL KD groups at p = 0.08 (df = 1). Two-way ANOVA shows significant difference in pMLC levels at p < 0.05 between control and E2-treated cells in the LKO group, but not in the EVL KD group. No significant differences in Arp2/3 levels were observed at p < 0.05. ** p < 0.01. c Maximum intensity projections of z-series of control and fulv-treated eGFP and eGFP-EVL MCF7 cells. Scale bar is 10 µm. Scale bar is 5 µm for inset showing eGFP-EVL and pMLC. Larger fields of view from the same group are given in Supplementary Fig. . d Quantification of leading edge Arp2/3 and pMLC. Data from three independent experiments ( n = 21 cells per group; mean ± s.e.m.). Three-way ANOVA shows significant differences between eGFP and eGFP-EVL groups at p < 0.0001 (df = 1). Two-way ANOVA shows significant difference in pMLC levels at p < 0.05 between control and fulv-treated cells in the eGFP group, but not in the eGFP-EVL group. No significant differences in Arp2/3 levels were observed at p < 0.05. * p < 0.05. e Analysis of EVL localization at SCABs by iPALM in MCF7 cells expressing mEos2-EVL. Left panels show single and merged images of F-actin staining and mEos2-EVL; boxes indicate ROIs. Arrows indicate en face orientation of orthogonal projections. Orthogonal projections of ROIs are shown with corresponding histogram highlighting z-localization of F-actin (red) and mEos2-EVL (cyan); horizontal axes represent molecular counts (mol. cts.) for mEos2-EVL (top) and F-actin (bottom). Merge image scale bar is 5 µm. Orthogonal projection scale bar is 150 nm. f Leading edge kymography in control and iRFP670-EVL (green) expressing MCF7 cells (top and bottom rows, respectively), with eGFP-Lifeact (black) and MLC-mRuby2 (magenta), before and after ROCK inhibitor (50 µM) treatment (Supplementary Movie ). Left panels are images from time-lapse series before and after treatment. Scale bar is 10 µm. Line shows leading edge location at which kymographs were registered. Inset shows EVL channel separately. Right panel shows kymograph. Vertical scale bar is 10 µm. Horizontal scale bar is 10 min. n.s. nonsignificant

Article Snippet: The actin cytoskeleton was stained with Alexa Fluor-conjugated phalloidin (Alexa Fluor 568-Phalloidin or Alexa Fluor 647-Phalloidin; Thermo Fisher) diluted to 2.5% in phosphate-buffered saline (PBS); nuclei were stained with Hoechst 33342 (Thermo Scientific); pMLC was immunolabeled using human anti-(Thr18/Ser19) pMLC 2 no. 3674 (Cell Signaling) at 1:100 dilution; Arp2/3 was immunolabeled using human anti-Arp2/3 clone no.13C9 (Millipore) at 1:200 dilution; EVL was immunolabeled using human anti-EVL (a gift from Frank Gertler) at 1:50 dilution.

Techniques: Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: The MARCKS Protein Plays a Critical Role in Phosphatidylinositol 4,5-Bisphosphate Metabolism and Directed Cell Movement in Vascular Endothelial Cells

doi: 10.1074/jbc.M110.196022

Figure Lengend Snippet: Insulin-promoted phosphorylation of N-WASP and Arp2/3 in cultured endothelial cells. Show are immunoblots from an insulin dose-response experiment probed with antibodies directed against the PIP2- and actin-binding proteins N-WASP (A and B) and Arp2/3 (C and D). Lysates prepared from insulin-treated BAEC were resolved by SDS-PAGE and analyzed in immunoblots probed with antibodies for total or phospho-N-WASP (phosphorylated residues pTyr-256 (pN-WASP(Tyr256) and pSer-484 (pN-WASP(Ser484), as shown) or total or phospho-Arp2/3 (pTyr-265). A and C, representative immunoblots. B and D, quantitative plots derived from pooled data. Each bar in the graphs represents the mean ± S.E. of four independent experiments that yielded similar results. *, p < 0.05; **, p < 0.01.

Article Snippet: Antibodies directed against Akt, phospho-Akt (Ser-473), pan-phospho-PKC, phospho-ERK1/2 (Thr-202/Tyr-204), and ERK1/2 were from Cell Signaling Technology, Inc. Polyclonal antibodies against phospho-N-WASP (Thr-237), phospho-N-WASP (Ser-484/Ser-485), N-WASP, phospho-Arp2/3 (Ser-265), and Arp2/3 were from ECM Biosciences ( 18 , 19 ).

Techniques: Cell Culture, Western Blot, Binding Assay, SDS Page, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: The MARCKS Protein Plays a Critical Role in Phosphatidylinositol 4,5-Bisphosphate Metabolism and Directed Cell Movement in Vascular Endothelial Cells

doi: 10.1074/jbc.M110.196022

Figure Lengend Snippet: Model for the role of MARCKS in insulin-modulated PIP2 mobilization. Shown is a model that integrates the findings in this study that have explored the pathways of insulin-mediated PIP2 mobilization. In resting endothelial cells (upper panel), MARCKS sequesters PIP2 in caveolae, and N-WASP and Arp2/3 are quiescent and not interacting with one another. Insulin receptor activation (lower panel) leads to the mobilization of PIP2 from MARCKS, accompanied by MARCKS phosphorylation and translocation from caveolae to intracellular sites. The mobilized PIP2 binds to and activates N-WASP and Arp2/3, leading to alterations in the actin cytoskeleton that are critical determinants of directed endothelial cell movement. See text for further discussion.

Article Snippet: Antibodies directed against Akt, phospho-Akt (Ser-473), pan-phospho-PKC, phospho-ERK1/2 (Thr-202/Tyr-204), and ERK1/2 were from Cell Signaling Technology, Inc. Polyclonal antibodies against phospho-N-WASP (Thr-237), phospho-N-WASP (Ser-484/Ser-485), N-WASP, phospho-Arp2/3 (Ser-265), and Arp2/3 were from ECM Biosciences ( 18 , 19 ).

Techniques: Activation Assay, Translocation Assay