anti shp2 ptpn11  (Boster Bio)

Journal: iScience

Article Title: Pulmonary surfactants and the respiratory-renal connection in steroid-sensitive nephrotic syndrome of childhood

doi: 10.1016/j.isci.2022.104694

Figure Lengend Snippet: Effect of steroids on SIRPα and podocyte activation (A) Co-immunoprecipitation between SIRPα and SHP-2 in cultured human podocytes incubated with serum from an age-matched control subject (C1) or from two different subjects with MCD (Pt1 and Pt2) in remission, relapse (Rl), or relapse in the presence of dexamethasone (100 nM) (Rl + Dex). (C) Co-immunoprecipitation between SIRPα and nephrin in cultured human podocytes incubated with serum from an age-matched control subject (C1) or or from two different subjects with MCD (Pt1 and Pt2) in remission, relapse (Rl), or relapse in the presence of dexamethasone (100 nM) (Rl + Dex). (D) Ratio of interaction between SHP-2 and SIRPα or nephrin in cultured human podocytes incubated with serum from three controls (black symbols) or six subjects with SSNS/MCD in remission (red symbols), relapse (blue symbols), or relapse and dexamethasone (purple symbols). D) Representative western blot for total and phosphorylated nephrin (P-Nephrin) expression in cultured human podocytes incubated with serum from a control (C1) subject or from a patient with MCD (Pt1) in remission (Rm), relapse (Rl), or relapse in the presence of dexamethasone (Rl + dex) for 8 h. (E) Densitometry analysis of P-Nephrin expression corrected for total nephrin following incubation with serum from three control subjects (black) or six patients with SSNS/MCD in remission, relapse, or relapse in the presence of dexamethasone for 8 h. Data presented are an average from three independent replicates for each subject and time point. (F) Confocal imaging of polymerized actin (F-actin, green) in cultured human podocytes incubated with serum from a patient with MCD (Pt1) in remission, relapse, or relapse in the presence of dexamethasone for 8 h. Size bars: 20 μm. (G) IL6 -top- and TNFa -bottom- mRNA expression in human cultured podocytes incubated for 24 h with serum from three age-matched control subjects (black symbols) or six patients with SSNS/MCD in remission (red symbols), relapse (blue symbols), or relapse in the presence of dexamethasone (Purple symbols). Data presented are an average from three independent replicates for each subject. (H) Proposed steroid-sensitive areas in podocytes. Steroids will have no effect on direct podocyte SIRPα activation by surfactants, the cytosolic release of SHP2 and the dephosphorylation of nephrin. However, steroids ameliorate the cytoskeletal depolymerization of actin and podocyte inflammation. Possibly, steroids may also act in vivo by reducing the surfactant release to the circulation after a pulmonary tract infection. Statistical Analysis: One-Way ANOVA, Tukey post hoc analysis. Data in panles (C) and (G) are represented as mean ± SEM.

Article Snippet: Anti human SHP2 (PTPN11) , Cell Signaling , 3752 RRID: AB_2300607.

Techniques: Activation Assay, Immunoprecipitation, Cell Culture, Incubation, Control, Western Blot, Expressing, Imaging, De-Phosphorylation Assay, In Vivo, Infection

Journal: iScience

Article Title: Pulmonary surfactants and the respiratory-renal connection in steroid-sensitive nephrotic syndrome of childhood

doi: 10.1016/j.isci.2022.104694

Figure Lengend Snippet:

Article Snippet: Anti human SHP2 (PTPN11) , Cell Signaling , 3752 RRID: AB_2300607.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: SAR of furanylbenzamide inhibitors and analogs

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Mechanism of action, inhibition, and binding studies of SHP2 inhibitors. A , Michaelis–Menten kinetic studies for the SHP2 inhibitor SBI-4668 with SHP2-E76K. Plots show the initial rates (V) at various substrates (DiFMUP) and inhibitor concentrations fitted to the Michaelis–Menten equation for noncompetitive inhibition. Relative fluorescence units per minute (RFU/min) are represented as mean ± SD (n = 3). B , Eadie–Hofstee plots of the Michaelis–Menten kinetic studies with compound SBI-4668 from A . C , dose–response curves for SBI-2130 with SHP2-E76K after various preincubation times of inhibitor with SHP2. RFU/min are represented as mean ± SD (n = 3). No time-dependent inhibition was observed as demonstrated by the similar potency for the various time points. D , dose–response curves for SBI-2130 with SHP2cat with or without a 10× inhibitor/protein preincubation and jump dilution. Identical IC 50 curves indicate that SBI-2130 is a reversible inhibitor. RFU/min are represented as mean ± SD (n = 4). E , dose-dependent binding of SBI-4668 to SHP2cat in a protein thermal shift (PTS) assay. Thermal stabilization of SHP2 by SBI-4668 is shown by the increase in the SHP2 melting temperature (Δ T m ) compared with vehicle control (DMSO). Δ T m values are represented as mean ± SD (n = 4). DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 domain–containing phosphatase 2; SHP2cat, SHP2 catalytic domain.

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Inhibition, Binding Assay, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Selectivity of furanylbenzamide inhibitors for SHP2 over related phosphatases PTP1B and STEP (IC 50 values in micromolar; each PTP construct was comprised of the catalytic domain)

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Construct

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Efficacy and selectivity of the SHP2 inhibitors in cellular cancer models and in patient-derived AML samples. A , viability of AML (Kasumi-1) and esophageal cancer (KYSE-520) cells in the presence of various concentrations of SHP2 active (SBI-2130, SBI-4668, and SBI-3404) and nonactive (SBI6339, negative control) furanylbenzamides after 3 days in culture. Cell viability is shown as a percentage of the vehicle (DMSO) control and represents the mean ± SD (n = 4). B , SBI-4668 dose–response curves in cell viability assays using Kasumi-1, KYSE-520, MOLM-13, and MV4-1 cells after 3 days in culture. The percentages compared with DMSO vehicle control were curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope) and represent the mean ± SD (n = 4). C , cell viability of BT-459 and MDA-MB-468 triple-negative breast cancer (TNBC) cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor SHP099 after 5 days in culture. Cell viability is shown as a percentage of DMSO vehicle control, representing mean ± SD (n = 2), and curve fitted as in B . D , colony formation assay (11 days) of BT-459 and MDA-MB-468 TNBC cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor SHP099. E , phospho-ERK1/2 (p-ERK1/2) immunoblot analysis from total cell lysates of MOLM-13 AML cells treated with SBI-4668 at the indicated concentrations or with SHP2 allosteric inhibitor RMC-4550 (RMC, 1 μM) for 3 h or 24 h. The quantitation of p-ERK1/2 levels is shown as the percentage of the DMSO (vehicle) control and represents data from three independent experiments (mean ± SD). F , viability of AML patient-derived cells in the presence of 10 μM SBI-4668 or 10 μM SHP2 allosteric inhibitor RMC-4550 after 2, 4, or 6 days in culture. Cell viability is shown as a percentage of the DMSO vehicle control and represents the mean ± SD (n = 4; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; unpaired t test with Welch's correction). G , viability of MOLM-13 cells and MOLM-13-Cas9-mCherry cells with SHP2 KO in the presence of SBI-2130, SBI-4668, and allosteric inhibitor SHP099 at various concentrations (10-point dose response). The percentages compared with the DMSO vehicle control were curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope) and represent the mean ± SD (n = 4). SHP2 protein levels in regular MOLM-13 cells (WT), MOLM-13-Cas9-mCherry cells (Cas9), and MOLM-13-Cas9-mCherry cells with SHP2 KO were evaluated by immunoblot analysis using SHP2 antibodies. AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 domain–containing phosphatase 2.

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Derivative Assay, Negative Control, Control, Colony Assay, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

doi: 10.1016/j.jbc.2021.101477

Figure Lengend Snippet: Evaluation of SBI-4668 in U-937 AML cells expressing the SHP2 oncogenic variant G60R. A , cell viability of U-937 AML cells in the presence of various concentrations of SBI-4668 or the SHP2 allosteric inhibitor RMC-4550 after 3 days in culture. Cell viability is shown as a percentage of the DMSO vehicle control, representing the mean ± SD (n = 4) and curve fitted using nonlinear regression (log[inhibitor] versus normalized response, variable slope). B , phospho-ERK1/2 (p-ERK1/2) immunoblot analysis from total cell lysates of U-937 AML cells treated with SBI-4668 at the indicated concentrations or with SHP2 allosteric inhibitor RMC-4550 (RMC, 1 μM) for 24 h. The quantitation of p-ERK1/2 levels is shown as the percentage of the DMSO (vehicle) control and represents data from three independent experiments (mean ± SD). AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 dom/ain–containing phosphatase 2.

Article Snippet: The following antibodies were used: Phospho-Erk1/2 Ab (catalog no.: 9101S; Cell Signaling); total Erk1/2 Ab (catalog no.: 9102S; Cell Signaling); SHP2 Ab (catalog no.: A301-544A; lot no.: 1; Bethyl); and GAPDH (14C10) mAb (catalog no.: 2118S; lot no.: 14; Cell Signaling).

Techniques: Expressing, Variant Assay, Control, Western Blot, Quantitation Assay

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