Figure 3.
Figure 3. Generation of stable transfectants inducibly expressing WT and R459M SHP-1 variants. ES cell transfectants were generated as described in “Materials and methods.” (A) Transfectants were cultured for 24 hours in the presence or absence of 500 ng/mL Tet, and lysates were prepared. Lysates were separated by SDS-PAGE and immunoblotted for SHP-1. WT clone 15 and R459M clone 20 are shown as examples. Arrows indicate the positions of the expressed and endogenous proteins. (B) Transfectants were cultured as in panel A, but the induction was extended for 96 hours. Lysates were taken after each 24-hour period. Lysates were separated by SDS-PAGE and immunoblotted with anti-myc epitope tag antibody (9E10) and then reprobed for SHP-1. R459M clone 20 is shown as an example. Arrows indicate the positions of the expressed and endogenous proteins. (C) Total cell extracts (T), NP40 soluble extracts comprising cytosolic and membrane proteins (C/M), and nuclear fractions (N) were prepared from 1 × 107 ES cells from WT clone 3 and R459M clone 48. For the total cell extracts, the equivalent of 1 × 105 cells was loaded per lane and the equivalent of 2 × 105 cells was loaded per lane for the cytosolic and nuclear fractions (ie, double the amount of total extract separated). Duplicate gels were run, and 7.5% acrylamide gels were probed with the anti–SHP-1 antibody (α-SHP-1) or the anti-gp130 antibody (α-gp130) and subsequently were stripped and reprobed with anti-myc tag or anti-STAT3 antibodies. The 10% acrylamide gel was probed for Oct-4 and then stripped and reprobed for SHP-2. (D) WT clone 3 and R459M clone 48 ES cell transfectants were plated in the presence and absence of 500 ng/mL Tet for 48 hours. Extracts from cells growing exponentially were prepared, and 1 mg cell extract (Pre IP) was used to generate SHP-2 immunoprecipitates (α-SHP2). Immunoblotting was performed with 4G10 (α-pY), and the blot was stripped and reprobed with anti–SHP-2 antibodies to control for loading and then probed for SHP-1 to demonstrate expression. Arrows indicate the molecular weights of tyrosine-phosphorylated proteins coprecipitating with SHP-2.

Generation of stable transfectants inducibly expressing WT and R459M SHP-1 variants. ES cell transfectants were generated as described in “Materials and methods.” (A) Transfectants were cultured for 24 hours in the presence or absence of 500 ng/mL Tet, and lysates were prepared. Lysates were separated by SDS-PAGE and immunoblotted for SHP-1. WT clone 15 and R459M clone 20 are shown as examples. Arrows indicate the positions of the expressed and endogenous proteins. (B) Transfectants were cultured as in panel A, but the induction was extended for 96 hours. Lysates were taken after each 24-hour period. Lysates were separated by SDS-PAGE and immunoblotted with anti-myc epitope tag antibody (9E10) and then reprobed for SHP-1. R459M clone 20 is shown as an example. Arrows indicate the positions of the expressed and endogenous proteins. (C) Total cell extracts (T), NP40 soluble extracts comprising cytosolic and membrane proteins (C/M), and nuclear fractions (N) were prepared from 1 × 107 ES cells from WT clone 3 and R459M clone 48. For the total cell extracts, the equivalent of 1 × 105 cells was loaded per lane and the equivalent of 2 × 105 cells was loaded per lane for the cytosolic and nuclear fractions (ie, double the amount of total extract separated). Duplicate gels were run, and 7.5% acrylamide gels were probed with the anti–SHP-1 antibody (α-SHP-1) or the anti-gp130 antibody (α-gp130) and subsequently were stripped and reprobed with anti-myc tag or anti-STAT3 antibodies. The 10% acrylamide gel was probed for Oct-4 and then stripped and reprobed for SHP-2. (D) WT clone 3 and R459M clone 48 ES cell transfectants were plated in the presence and absence of 500 ng/mL Tet for 48 hours. Extracts from cells growing exponentially were prepared, and 1 mg cell extract (Pre IP) was used to generate SHP-2 immunoprecipitates (α-SHP2). Immunoblotting was performed with 4G10 (α-pY), and the blot was stripped and reprobed with anti–SHP-2 antibodies to control for loading and then probed for SHP-1 to demonstrate expression. Arrows indicate the molecular weights of tyrosine-phosphorylated proteins coprecipitating with SHP-2.

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