Journal: Scientific reports
Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells.
doi: 10.1038/s41598-025-98266-8
Figure Lengend Snippet: Fig. 5. Rap1 overexpression reverses the expression of key proteins in CD147 knockdown mediated cell proliferation, apoptosis and EMT. (A, B) Western blot analysis of Rap1 and Rap1GAP protein levels in HCT116 and SW620 cells after CD147 knockdown (shCD147). β-actin served as a loading control. Data represent mean ± SD (n = 3; **P < 0.01, ***P < 0.001 vs. shNC, Student’s t-test). Original blots are presented in Supplementary Fig. 3. (C) qRT-PCR validation of Rap1 mRNA overexpression in CD147-knockdown cells. Expression normalized to β-actin (n = 3; ***P < 0.001 vs. HCT116 or SW620, Student’s t-test). (D, E) Western blot analysis of c-Myc, Bcl-2, Bax, N-cadherin, and E-cadherin protein levels in CD147-Knockdown cells with Rap1 overexpression (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. shNC, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. shCD147, one-way ANOVA). Original blots are presented in Supplementary Fig. 4.
Article Snippet: Supplier Antibody dilution used WB/IF Anti CD147 mouse monoclonal antibody 66443-1-AP ProteinTech Group, Inc. 1:5000 /1:200 Anti N-cadherin rabbit polyclonal antibody 22018-1-AP ProteinTech Group, Inc. 1:5000 Anti E-cadherin rabbit polyclonal antibody 20874-1-AP ProteinTech Group, Inc. 1:10000 Anti c-myc mouse monoclonal antibody 67,447-AP ProteinTech Group, Inc. 1:5000 Anti RAP1GAP rabbit polyclonal antibody 19174-1-AP ProteinTech Group, Inc. 1:2000 Anti RAP1 rabbit polyclonal antibody 14595-1-AP ProteinTech Group, Inc. 1:1000 Anti β-actin rabbit polyclonal antibody 20536-1-AP ProteinTech Group, Inc. 1:8000 HRP-conjugated Goat Anti-Mouse IgG(H + L) SA00001-1 ProteinTech Group, Inc. 1:10000 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) ab150116 Abcam 1:200 Table 1.
Techniques: Over Expression, Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Biomarker Discovery
Proteintech is a verified supplier 
![Overexpression of SHANK3 blocks Rap1 activation induced by talin recruitment to the plasma membrane. (A–C) SHANK3 tagged with Myc–mAzurite was transfected into A5 CHO cells stably expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells triple positive for mAzurite, GFP and mCherry were sorted by flow cytometry. Cells expressing Myc–mAzurite without SHANK3 served as a control. (A) Western blot analysis of SHANK3–Myc–mAzurite expression in these cells. β-actin served as a loading control. SHANK3 overexpression did not affect the levels of CRY2–mCherry–talin. The images represent two independent experiments. (B) SHANK3 overexpression inhibits Rap1 activation in response to the optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of five experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (C) SHANK3 blunts activation of integrin αIIbβ3 in response to the optogenetic recruitment of talin to the plasma membrane. Activation of integrin αIIbβ3 was monitored by flow cytometry using the PAC-1 antibody and expressed as the fold increase relative to that observed when cells were maintained in the dark. Data represent means±s.e.m. of five experiments (* P <0.05; paired two-tailed Student's t -test). (D–G) Lentiviruses encoding the WT SPN domain of SHANK3 [mAzurite–FLAG–SPN (WT)], the R12C SPN mutant [mAzurite–FLAG–SPN (R12C)] or the L68P SPN mutant [Myc–mAzurite–FLAG–SPN (L68P)] were transduced into immortalized murine lung endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells infected with empty lentiviral vector served as controls. (D) WT SPN, but not R12C or L68P SPN, inhibits Rap1 activation following optogenetic recruitment of talin to the plasma membrane. Data represent mean±s.e.m. of four experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). (E) WT SPN, but not the R12C or L68P SPN mutants, inhibits specific fibrinogen binding to integrin αVβ3 upon optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of eight experiments (N.S., not significant; * P <0.05; ** P <0.01; paired two-tailed Student's t -test). (F,G) Duolink proximity ligation assay (PLA) was performed to examine the effects of SHANK3 SPN on the association of Rap1 with CRY2–mCherry–talin in endothelial cells. (F) Schematic representation of the Duolink PLA. Created in BioRender by Liao, Z., 2025. https://BioRender.com/m47c469 . This figure was sublicensed under CC-BY 4.0 terms. (G) After 30 min of incubation at room temperature in the absence or presence of blue light illumination, cells were fixed, permeabilized and stained with rabbit anti-mCherry and mouse anti-Rap1 antibodies. Then, Duolink PLA flow cytometry was performed to assess the interaction between CRY2–mCherry–talin and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8058/pmc11928058/pmc11928058__joces-138-263595-g3.jpg)
