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. 2023 Dec 15;7:136. doi: 10.1038/s41698-023-00486-6

Fig. 2. SHP2 regulates TGFβ activity.

Fig. 2

a TGFβ responsive luciferase (CAGA luciferase) of HEK293T cells transfected with two independent hairpins targeting SHP2 or a hairpin targeting GFP were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Error bars represent S.D. of triplicates. Experiments are representative of three independent experiments. **P < 0.01, ***P < 0.001 as determined by Student’s t-test. b TGFβ responsive luciferase (CAGA luciferase) of HEK293T cells transfected with pcDNA (Ctl), wildtype SHP2, catalytically active SHP2 (E76A), and catalytically inactive mutant of SHP2 (C459S) were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Error bars represent S.D. of triplicates. Experiments are representative of three independent experiments. *P < 0.05 as determined by Student’s t-test. c HEK293T cells transfected with siRNA targeting SHP2 or relevant control and treated overnight with TGFβ (100 pM). Immunoblotted lysates are probed with indicated antibodies. d Quantification of c comparing phospho-SMAD2 to corresponding SMAD2. Density was evaluated with IMAGE J. Bars represent mean ± S.D. from three independent experiments. A two-tailed Student’s t-test compares the treated populations. *P < 0.05. e HEK293T cells transfected with SHP2 or relevant control and treated overnight with TGFβ (100 pM). Immunoblotted lysates were probed with indicated antibodies. f Quantification of e comparing phospho-SMAD2 to corresponding SMAD2. Density was evaluated with IMAGE J. Bars represent mean ± S.D. from three independent experiments. A two-tailed Students’ t-test compares the treated populations. *P < 0.05. g NBT-II cells treated with either TGFβ (100 pM) or SHP099 (1 or 5 μM) or both for 24 or 48 h. Lysates were probed with indicated antibodies. h Quantification of g comparing phospho-SMAD2 to corresponding SMAD2. Density was evaluated with IMAGE J. Bars represent mean ± S.D. from three independent experiments. A two-tailed Student’s t-test compares the treated populations. *P < 0.05. i TGFβ responsive luciferase (CAGA luciferase) of NBT-II cells stimulated overnight with TGFβ (100 pM) or SHP099 (5 μM) or in combination. Lysates were collected and luciferase measured by a luminometer. Error bars represent S.D. of triplicates. Experiments are representative of three independent experiments. **P < 0.01 as determined by Student’s t-test. j TGFβ responsive luciferase (CAGA luciferase) of HEK293T cells transfected with shSHP2 or a hairpin targeting GFP were stimulated where indicated with BMP7 (50 μg/μl) overnight before lysis. Error bars represent S.D. of triplicates. Experiments are representative of three independent experiments. ****P < 0.0001 as determined by Student’s t-test.