Journal: Biochemical Journal

Article Title: Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

doi: 10.1042/BJ20121750

Figure Lengend Snippet: ( A ) Co-occurrence matrix for the phosphoproteomic dataset. Co-occurrences are calculated by counting the number of times any two phosphopeptides cluster together across all 216 clustering sets. The matrix is then normalized as a percentage of the number of times they cluster out of 216 times and subsequently clustered using hierarchical clustering with Ward linkage. The first seven clusters that display higher-than-average co-occurrence are highlighted by the red box. ( B ) The co-occurrence map of the first seven clusters expanded from ( A ). Cluster 1 contains DDR2 Tyr 740 as well as SHP2 Tyr 62 . Cluster 2 contains three DDR2 phosphopeptides containing Tyr 736 , Tyr 736 /Tyr 740 and Tyr 618 . Cluster 3 contains DDR2 Tyr 813 . ( C ) The dynamics of the members of each of the three DDR2 phosphorylation site-containing robust clusters. ( D ) Distribution of co-occurrences in the ensemble clustering result. A single hard clustering from the ensemble is obtained by cutting the Ward-linked hierarchically clustered co-occurrence matrix in (A) into 40 clusters. The co-occurrences within every cluster is then plotted to give an idea of the distribution. Comparison of phosphotyrosine/phosphotyrosine (pY/pY) sites are in red, phosphoserine/phosphothreonine (pS/pT):phosphoserine/phosphothreonine (pS/pT) are in blue, and cyan is of the co-occurrence events between phosphotyrosine (pY) and phosphoserine/phosphothreonine (pS/pT) sites.

Article Snippet: Following one-dimensional separation and transfer on to PVDF membrane, the membrane was incubated overnight at 4°C with 1:1000 dilutions of goat anti-DDR2 (R&D Systems), mouse anti-phosphotyrosine [4G10 (Millipore) or PY100 (Cell Signaling Technology)] or rabbit anti-SHP2 (Santa Cruz Biotechnology) antibodies, a 1:500 dilution of a rabbit anti-(SHP2 p-Tyr 542 ) (Cell Signaling Technology) antibody or with a 1:5000 dilution of a mouse anti-α-tubulin (Sigma).

Techniques: Phospho-proteomics, Comparison

Journal: Cell Death & Disease

Article Title: Cryptotanshinone inhibits human glioma cell proliferation in vitro and in vivo through SHP-2-dependent inhibition of STAT3 activation

doi: 10.1038/cddis.2017.174

Figure Lengend Snippet: Inhibitory effect of CTS on p-STAT3 is mediated by SHP-2. ( a ) U251 cells were treated with pervanadate at indicated concentration for 1h, and with additional 10 μ M CTS for 2 h. Proteins were analyzed by western blotting with STAT3, p-STAT3 (Tyr705) and GAPDH antibodies. (PI: phosphoesterase inhibitors, pervanadate). ( b ) U251 cells were treated with 10 μ M CTS for 0, 0.15, 0.5, 1, 2, 4 h. Proteins were analyzed by western blotting with SHP-1, TC-PTP, SHP-2, p-SHP-2 (Tyr542), p-SHP-2 (Tyr580) and GAPDH antibodies. ( c – e ) U251 cells were transfected with specific phosphates siRNA including SHP-1 ( c ), TC-PTP ( d ), SHP-2 ( e ) or negative control (NC) for 48 h, and then treated with CTS with the indicated concentration for 120 min, followed by western blot analysis with the indicated phosphates antibodies. Expression of p-STAT3 (Tyr705) relative to STAT3 were quantified by densitometry. Results are the mean±S.E.M. ( n =3 for each group). ## P <0.01, ### P <0.001 versus control. ( f ) Cell lysates from U251 cells treated with 0, 5, 10 μ M CTS for 2 h were subjected to SHP-2 phosphatase activity assay. Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01 versus CTS 0 μ M group. ( g and h ) U251 cells were pretreated with 25 μ M NSC-87877 for 30 min, followed by 10 μ M CTS treatment for 120 min. Total cell lysates were prepared and subjected to western blot analysis with indicated antibodies ( g ) or SHP-2 phosphatase activity assay ( h ). Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01, versus CTS 0 μ M group

Article Snippet: Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).

Techniques: Concentration Assay, Western Blot, Transfection, Negative Control, Expressing, Phosphatase Assay

Journal: Cell Death & Disease

Article Title: Cryptotanshinone inhibits human glioma cell proliferation in vitro and in vivo through SHP-2-dependent inhibition of STAT3 activation

doi: 10.1038/cddis.2017.174

Figure Lengend Snippet: Growth inhibition of intracranial tumor by CTS treatment extended survival of nude mice bearing intracerebral U87 xenografts treated with CTS. ( a ) Growth inhibition of intracranial tumor by CTS treatment. Left: after hematoxylin–eosin staining, tumor size was determined by the areas that were measured at the maximal brain tumor dimensions in the coronal sections. The red circles indicate tumor tissue. Scale bar, 1 mm. Data were calculated by taking the tumor size of control as 100%. Data are expressed as mean±S.E.M., n =5 for each group. * P <0.05; ** P <0.01 versus Model control group. Right: higher magnification in HE staining. Scale bar, 50 μ m. ( b ) After xenografts for 7 days, nude mice were treated with CTS from day 8 to 21 and were observed after discontinuation of therapy. Statistical significance was achieved by Cox–Mantel and Wilcoxon analyses of a Kaplan–Meier survival curve ( n =5). ( c ) Some samples from the harvested tumor tissue on Day 28 were used in detecting tyrosine phosphatase activity of SHP-2. Data are expressed as mean±S.E.M., n =3 for each group. * P <0.05; ** P <0.01, versus Model control group

Article Snippet: Primary antibodies were used as follows: antibodies against SHP-1 and SHP-2 (Epitomics, Burlingame, CA, USA); STAT3, p-STAT3 (Tyr705), p-STAT3 (Ser727), p-p44/42 Erk1/2 (Thr202/Tyr204), cyclin D1, cyclin E1, survivin, p-SHP-2 (Tyr580), p-SHP-2 (Tyr542) and Histon H3 (Cell Signaling Technology); antibody against Ki67 (BD Bioscience); antibodies against TC-PTP, SHP-2, Mcl-1 (Proteintech, Wuhan, Hubei, China); GAPDH (Beyotime).

Techniques: Inhibition, Staining, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Exerts Anticancer Effects on Pituitary Adenoma Progression via Inducing Cell Apoptosis and Inhibiting Cell Migration

doi: 10.3390/ijms23052664

Figure Lengend Snippet: TAM activated TLR4 signaling and inactivated immune checkpoint SHP1/SHP2. ( A ) The protein expression levels of the TLR4/NF-κB signaling pathway normalized to the GAPDH in RAW 264.7 cells were detected. ( B ) The expression levels of SIRPα protein, the phosphorylation levels of SHP1 and SHP2 proteins in RAW 264.7 cells were detected using Western blot. ( C , D ) The expression levels of SIRPα protein, the phosphorylation levels of SHP1 and SHP2 proteins in GH3 and AtT-20 cells were detected. ( E ) The expression levels of SIRPα protein, the activity of SHP1 and SHP2 proteins in tumor tissues were also detected. Bars indicates SEM, VC vehicle control, refers to tumor-bearing mice treated with the vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control or VC. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cells treated with IL-TAM: tamoxifen.

Article Snippet: Western blotting was performed as per standard protocols, and the membranes were probed with antibodies targeting p-JAK1 (74129, Rt, 1:500), JAK1 (29261, Rt, 1:1000), p-STAT6 (56554, Rt, 1:500), p-PI3K (17366, Rt, 1:500), PI3K (4249, Rt, 1:1000), p-AKT (4060, Rt, 1:500), AKT (4685, Rt, 1:1000), p53 (48818, Ms, 1:1000), Bax (14796, Rt, 1:1000), p-NF-κB p65 (3033, Rt, 1:500), NF-κB p65 (8242, Rt, 1:1000), p-IκBα (2859, Rt, 1:500), IκBα (4814, Ms, 1:1000), p-IKKα/β (2697, Rt, 1:500), IKKα (11930, Ms, 1:1000), IKKβ (8943, Rt, 1:1000), p-SHP1 (8849, Rt, 1:500), SHP1 (26516, Rt, 1:1000), p-SHP2 (5431, Rt, 1:500), SHP2 (3397, Rt, 1:1000), SIRPα (47027, Rt, 1:1000) (Cell Signaling, Danvers, MA, USA), STAT6 (YT4454, Rt, 1:1000), MyD88 (YM33092, Ms, 1:1000) (Immunway, Plano, MA, USA), GAPDH (10494-1-AP, Ms, 1:1000), INOS (22226-1-AP, Rt, 1:1000) (Proteintech, Wuhan, Hubei, China), Bcl-2 (ab32124, Rt, 1:1000) (Abcam, Cambridge, MA, USA) and TLR4 (sc-293072, Ms, 1:1000) (santa cruz biotechnology, Silicon Valley, CA, USA)positive bands were detected using an ECL Kit (CWBIO, Beijing, China) and the Tanon Chemiluminescence Image Analysis System (Shanghai, China).

Techniques: Expressing, Phospho-proteomics, Western Blot, Activity Assay, Control