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: PLoS Pathogens

Article Title: HTLV-1 bZIP Factor Enhances T-Cell Proliferation by Impeding the Suppressive Signaling of Co-inhibitory Receptors

doi: 10.1371/journal.ppat.1006120

Figure Lengend Snippet: (A) Phosphorylation levels of SHP-2 (Tyr580) in CD4 + T cells of non-Tg and HBZ-Tg mice were analyzed by flow cytometry. Splenocytes of non-Tg or HBZ-Tg mice (8 weeks old) were stained with anti-CD4 and pSHP-2 (Tyr580) antibodies. (B) The phosphorylation level of SHP-2 (Tyr580) of HBZ-transduced murine primary CD4 + T cells was analyzed by flow cytometry. After HBZ transduction, cells were stimulated with anti-CD3/PD-L1.Fc-coated beads at bead-to-cell ratio of 1:1 for 6 hours. (C-E) Dephosphorylation of ZAP-70, CD3ζ and PKCθ was analyzed by immunoblotting (left). Jurkat-mock and Jurkat-HBZ cells were stimulated with 20 mM H 2 O 2 or 0.5 mM pervanadate for 0, 5, 15, 30 or 45 min. Phosphorylation levels were also analyzed by densitometry (right). Relative protein levels were calculated as the ratio of phosphorylated protein to total protein.

Article Snippet: Anti-phospho-SHP-2 (Tyr580) antibody was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Flow Cytometry, Staining, Transduction, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques:

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log2 FC) and blue indicating an increase (2 log2 FC) in protein abundance.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition

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