ha sos1  (Addgene inc)

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: ECM1 recruits GRB2 and SOS1 to the membrane to activate the MAPK signaling pathway. A) Affinity purification MS analysis of ECM1 interaction complexes in C4‐2B cells (left). Representative mass spectra of GRB2 and SOS1 peptides (right). B) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells with ECM1 (200 ng mL −1 ) treatment. C) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with Veh (PBS) or ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D,E) WB analysis of GRB2 and SOS1 protein expression in whole lysis (WL) and membrane proteins from C4‐2B cells treated with increasing concentrations of ECM1 (0, 200, 400, 800 ng mL −1 ), or with the addition of either DMEM or CM. GAPDH was used as a loading control for whole lysis, and PMCA1 for membrane proteins. F) IP detection of the interaction between ECM1 and GRB2 as well as SOS1 in C4‐2B cells treated with CM. G) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with DMEM or CM. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). H,I) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Membrane, Affinity Purification, Staining, Expressing, Lysis, Control

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Phosphorylated ENO1 bridges ECM1 with GRB2 and SOS1 at the membrane. A) Representative mass spectra of ENO1 peptide. B) IP analysis detected the interaction between ECM1 and ENO1 in C4‐2B cells. C) IF staining and quantification of ENO1 and ECM1 in C4‐2B cells. Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). D) Proximity ligation assay (PLA) analysis of the interaction between ECM1‐Flag and ENO1 (Scale bar, 10 µm). E) IP assays of the interaction between ENO1 and GRB2 as well as SOS1 in C4‐2B cells in the presence or absence of ECM1 (200 ng mL −1 ). F) IP analysis detected the interaction between ECM1 and GRB2 as well as SOS1 in the indicated C4‐2B cells treated with ECM1 (200 ng mL −1 ). G) Expression of GRB2 and SOS1 protein in whole lysis and membrane proteins from the indicated C4‐2B cells with ECM1 (200 ng mL −1 ) treatment by WB analysis. H) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). I) Representative peptides showing phosphorylation of ENO1 at the Y189 site by affinity purification MS analysis of protein modifications in ECM1‐treated C4‐2B cells. J) Tyr phosphorylation detection of the immunoprecipitated ENO1 in indicated C4‐2B and LNCap cells with ECM1 treatment (200 ng mL −1 ). K) Tyr phosphorylation levels of HA‐ENO1‐WT and ‐Y189F in C4‐2B cells with or without ECM1 (200 ng mL −1 ) treatment. L) IF staining and quantification of GRB2, SOS1, and DiI in the indicated C4‐2B cells supplemented with ECM1 (200 ng mL −1 ). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). M,N) IP assays of the interaction between ECM1 and ENO1, GRB2 as well as SOS1 in the indicated C4‐2B cells in the presence of ECM1 (200 ng mL −1 ). O) WB analysis of MEK, p‐MEK, ERK1/2, and p‐ERK1/2 expression in the indicated C4‐2B cells treated with or without ECM1 (200 ng mL −1 ). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Membrane, Staining, Proximity Ligation Assay, Expressing, Lysis, Phospho-proteomics, Affinity Purification, Immunoprecipitation

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: PhAH attenuates ENO1‐mediated PCa cell resistance to ENZ. A) Chemical structure of PhAH. B) Ligand interaction diagram (left) and binding amino acid residue sites (right) of the highest‐scoring PhAH and ENO1 protein molecular docking complex (RMSD = 1.9977; E_score = −3.9538). C) WB detection of the interaction between ECM1 and ENO1, as well as Tyr phosphorylation of ENO1 following immunoprecipitating ENO1 in C4‐2B cells in the presence of ECM1 (200 ng mL −1 ) with or without PhAH (1 µM). D) IF staining and quantification of GRB2, SOS1, and DiI in C4‐2B cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). Pearson R value greater than 0.5 indicated co‐localization of the two proteins (Scale bar, 5 µm). E) Representative images (top) and quantification (bottom) of surviving colonies formed by C4‐2B and LNCaP cells treated with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM). F) Flow cytometry analysis showing representative images (top) and quantification (bottom) of apoptosis in C4‐2B and LNCaP cells with or without ECM1 (200 ng mL −1 ) or PhAH (1 µM) treatment. G) Representative BLI of intratibial tumors in mice treated daily oral treatment with Veh, ENZ (20 mg kg −1 ), ENZ (20 mg kg −1 ) combined with intraperitoneal (i.p.) injection of either Veh (DMSO) or PhAH (5 mg kg −1 ) twice weekly at 4 weeks before treatment, and at weeks 0, 6, and 8 during treatment (n = 6 per group). H) Quantification of BLI signals in intratibial tumors of mice before and after Tx as grouped in G (n = 6 per group). I) Representative micro‐CT images of intratibial lesions from mice after 8 weeks of treatment grouped as shown in G (arrows and circles indicate osteoblastic lesions, n = 6 per group). J) Representative H&E images of intratibial tumors grouped as shown in G (T, tumor; N, the adjacent non‐tumor tissues. Scale bars, 500 µm and 100 µm). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Binding Assay, Residue, Phospho-proteomics, Staining, Flow Cytometry, Injection, Micro-CT

Journal: Advanced Science

Article Title: Osteoblast‐Derived ECM1 Promotes Anti‐Androgen Resistance in Bone Metastatic Prostate Cancer

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Schematic diagram illustrating that increased osteoblast‐derived ECM1 from the bone microenvironment of BMPC patients induced by ENZ treatment, interacts with the ENO1 receptor on the prostate cancer cell membrane, further recruiting adapter proteins including GRB2 and SOS1, which activates the downstream MAPK signaling pathway to promote the proliferation of PCa cells and induce anti‐androgen resistance.

Article Snippet: [ ] The primary antibodies utilized for protein detection included ENO1 Rabbit pAb (Proteintech; 11204‐1‐AP, 1:2000), ECM1 Rabbit pAb (Proteintech; 11521‐1‐AP, 1:1000), GRB2 Rabbit mAb (Abcam; ab32037, 1:5000), SOS1 Rabbit pAb (Proteintech; 55041‐1‐AP, 1:1000), HA‐Tag Rabbit mAb (Cell Signaling Technology; #3724, 1:1000), His‐Tag Rabbit mAb (Cell Signaling Technology; #2365, 1:1000), Flag‐Tag Rabbit mAb (Cell Signaling Technology; #14793, 1:1000), MEK Rabbit mAb (Cell Signaling Technology; #8727, 1:1000), Phospho‐MEK (Ser217/Ser221) Rabbit mAb (Cell Signaling Technology; #9154, 1:1000), ERK1/2 Rabbit mAb (Cell Signaling Technology; #4695, 1:1000), Phospho‐ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho‐EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho‐IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325‐1‐Ig, 1:1000), Phospho‐FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06–1433, 1:1000) and Phospho‐Tyrosine Mouse mAb (P‐Tyr‐100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Derivative Assay, Membrane