Journal: PeerJ

Article Title: CDCP1 knockdown suppresses PDGFRβ/AKT pathway-mediated vascular smooth muscle cell proliferation by inhibiting PDGFRβ endocytosis

doi: 10.7717/peerj.19114

Figure Lengend Snippet: (A) Differential genes analysis after CDCP1 knockdown based on RNA-seq. (B) GO enrichment analysis of differential genes.(C) KEGG enrichment analysis of differential genes. (D) Western blot was used to detect the phosphorylation level of PDGFRβ and AKT under PDGF-BB stimulation at indicated times (0, 5, 15, 30 min). (E–I) Quantitative analysis of CDCP1 (E), PDGFRβ (F), phosphate-PDGFRβ (G), AKT (H), phosphate-AKT (I). Data were expressed as mean ± SD. * P < 0.05 indicated a statistically significant difference. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

Article Snippet: The membranes that were blotted underwent a blocking procedure using 5% non-fat milk for a duration of 1 h. Following this, they were incubated at 4 °C overnight with primary antibodies targeting CDCP1 (Cat. no. 12754-1-AP; Proteintech, Wuhan, China), Rab5 (Cat. no. 46449; Cell Signaling Technology, Danvers, MA, USA), PDGFRβ (Cat. no. ET1605-20; Huaan, Hangzhou, China), AKT (Cat. no. ET1609-51; Huaan, Hangzhou, China), p-PDGFRβ (Cat. no. 3166; Cell Signaling Technology, Danvers, MA, USA) and P-AKT (Cat. no. AF0016; Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Knockdown, RNA Sequencing, Western Blot, Phospho-proteomics

Journal: PeerJ

Article Title: CDCP1 knockdown suppresses PDGFRβ/AKT pathway-mediated vascular smooth muscle cell proliferation by inhibiting PDGFRβ endocytosis

doi: 10.7717/peerj.19114

Figure Lengend Snippet: (A) Co-immunoprecipitation assay was used to demonstrat the interaction between PDGFβ and NEDD4 after CDCP1 knockdown. (B) Quantitative analysis of NEDD4. (C) Cells were treated with 20 ng/mL PDGF-BB for 30 min, and stained. Proteins were detected with PDGFRβ (red) and NEDD4 (green) antibodies. (D) Quantitative analysis of confocal/NEDD4 (green). Data were expressed as mean ± SD. * P < 0.05 indicated a statistically significant difference. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

Article Snippet: The membranes that were blotted underwent a blocking procedure using 5% non-fat milk for a duration of 1 h. Following this, they were incubated at 4 °C overnight with primary antibodies targeting CDCP1 (Cat. no. 12754-1-AP; Proteintech, Wuhan, China), Rab5 (Cat. no. 46449; Cell Signaling Technology, Danvers, MA, USA), PDGFRβ (Cat. no. ET1605-20; Huaan, Hangzhou, China), AKT (Cat. no. ET1609-51; Huaan, Hangzhou, China), p-PDGFRβ (Cat. no. 3166; Cell Signaling Technology, Danvers, MA, USA) and P-AKT (Cat. no. AF0016; Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Co-Immunoprecipitation Assay, Knockdown, Staining

Journal: PeerJ

Article Title: CDCP1 knockdown suppresses PDGFRβ/AKT pathway-mediated vascular smooth muscle cell proliferation by inhibiting PDGFRβ endocytosis

doi: 10.7717/peerj.19114

Figure Lengend Snippet: (A and C) Cells were treated with 20 ng/mL PDGF-BB for 30 min, and stained. The relationship between PDGFRβ (red) and Clathrin (green), PDGFRβ (red) and Rab5 (green) was measured by IF. (B and D) Quantitative analysis of confocal/Clathrin (B) and confocal/Rab5 (D). (E) Co-immunoprecipitation assay was used to detect the relationship of PDGFRβ with Clathrin and Rab5 after CDCP1 knockdown. (F and G) Quantitative analysis of Clathrin (F) and Rab5 (G). Data are expressed as mean ± SD. * P < 0.05 indicated a statistically significant difference. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

Article Snippet: The membranes that were blotted underwent a blocking procedure using 5% non-fat milk for a duration of 1 h. Following this, they were incubated at 4 °C overnight with primary antibodies targeting CDCP1 (Cat. no. 12754-1-AP; Proteintech, Wuhan, China), Rab5 (Cat. no. 46449; Cell Signaling Technology, Danvers, MA, USA), PDGFRβ (Cat. no. ET1605-20; Huaan, Hangzhou, China), AKT (Cat. no. ET1609-51; Huaan, Hangzhou, China), p-PDGFRβ (Cat. no. 3166; Cell Signaling Technology, Danvers, MA, USA) and P-AKT (Cat. no. AF0016; Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Staining, Co-Immunoprecipitation Assay, Knockdown