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