Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: DDR2 expression is regulated by HSP47. A, spearman correlation analysis showed that mRNA levels of HSP47 and DDR2 were significantly correlated in human breast cancer tissues (TCGA; n = 532). B and C, silencing HSP47 significantly reduced DDR2 protein levels in MDA-MB-231 cells. Western blotting data were quantified by ChemImage (C), and DDR2 levels in HSP47-silenced cells (shA3) were normalized to control (shctrl; n = 3). Displayed are the means with S.E. **, p < 0.01, two-tailed Student's t test. D, total DDR2 and Tyr phosphorylation of DDR2 were assessed after DDR2 was immunoprecipitated from control and HSP47-silenced cells.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: Expressing, Western Blot, Two Tailed Test, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: DDR2 exhibits low stability in HSP47 silenced cells. A, DDR2 mRNA levels were measured by real-time RT-PCR in control and HSP47-silenced MDA-MB-231 cells. n.s., no significance. B, DDR2 stability was analyzed by Western blotting in control cells (shctrl) and HSP47-silenced (shA3) cells treated with cycloheximide (CHX, 100 μg/ml) at 0, 2, 4, and 8 h. C, quantification for B. **, p < 0.01, two-tailed Student's t test (n = 3). Displayed are the means ± S.D.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: HSP47 binds to DDR2 at the DS and DS-like domains. A, co-IP analysis was performed in HEK293 cells transfected with DDR2 and/or FLAG-HSP47 expression constructs. Protein complexes were pulled down with M2 beads and analyzed by Western blotting. B, co-IP analysis was performed in HEK293 expressing FLAG-DDR2 and/or HSP47. C, endogenous co-IP was performed in HS578 cells. Protein complexes were pulled down with HSP47 antibody and protein A/G beads. D, confocal microscopy imaging analyzing localization of HSP47 (green) and DDR2 (red) in HEK 293 cells. Nuclei was staining with (DAPI; blue). E, the cartoon showing the DDR2 protein structure. F, co-IP analysis was performed in HEK 293 cells expressing FLAG-HSP47 and truncated DDR2 (EX-domain and In-Domain). IB, immunoblotting.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Construct, Western Blot, Confocal Microscopy, Imaging, Staining

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: HSP47 expression prolongs membrane localization of DDR2 protein. A, a scheme showing how the DDR2–Dendra fused protein was photoconverted with 405-nm light. B, DDR2–Dendra2 expressing HEK293 cells were photoconverted within 5–10 s, and images were taken with TIRF microcopy under green and red channels immediately after photoconversion. C, TIRF images showed the membrane localization of DDR2–Dendra2 expressing in control and HSP47-silenced HEK293 cells. D, the red fluorescence intensity was quantified and normalized with ImageJ; the live cell images were taken with TIRF microcopy every 10 min after photoconversion. The experiment was repeated twice, and the fluorescence intensity was quantified in total 50 cells. Displayed are the means ± S.E.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: DDR2 rescues cell migration in HSP47-silenced MDA-MB 231 cells. A, single-cell migration was performed in DDR2 overexpression or not MDA-MB-231 shctrl cells and MDA-MB-231 shA3 cells for 8 h. The migration pathway were analyzed by Excel with 50 single cells for each group. B and C, quantification analysis of single cells' pathway length and distance from the origin for each group (n = 50). Displayed are the means ± S.E. ***, p < 0.0001, one-way ANOVA test.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: Migration, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Heat shock protein 47 (HSP47) binds to discoidin domain–containing receptor 2 (DDR2) and regulates its protein stability

doi: 10.1074/jbc.RA119.009312

Figure Lengend Snippet: DDR2 rescues cell invasion in HSP47-silenced MDA-MB-231 cells. A and B, phase images and quantification data showing the invasive growth of control and HSP47-silenced MDA-MB-231 cells in the presence or absence of exogenous DDR2 in 3D culture (n = 11). Displayed are the means ± S.E. ***, p < 0.0001, one-way ANOVA test. The 3D culture assay was repeated twice. C and D, phase images and quantification of cancer cell invasion in Transwell analysis. Control and HSP47-silenced MDA-MB-231 cells in the presence or absence of exogenous DDR2 were plated on Matrigel-coated Transwell and invaded cells were quantified under phase contrast microcopy (n = 10). Displayed are the means ± S.E. ***, p < 0.0001, one-way ANOVA test.

Article Snippet: Pierce glycoprotein staining kit was purchased from Thermo Scientific (24562) Antibodies against the following proteins were obtained from the following companies: DDR2 (R&D, AF2538) and HSP47 (Santa Cruz, sc-5293).

Techniques: