MASL1 physically interacts with Raf1. (A) 239T cells were transfected with myc-DDK-tagged MASL1-pCMV6-Entry plasmid and collected for protein lysates 48 hours after transfection. The protein lysates were subjected to pull-down with GST-Raf1-RBD coupled to glutathione resin and the bound proteins were analyzed by western blot using anti-MASL1 antibody (top). (Lane 1), mock-transfected 293T cell lysate pulled-down with GST-Raf1-RBD (Mock). (Lane 2), control vector-transfected 293T cell lysate pulled-down with GST-Raf1-RBD (Control pCMV6). (Lane 3), myc-DDK-tagged MASL1-transfected 293T cell lysate pulled-down with GST-Raf1-RBD (MASL1 pCMV6). (Lane 4), GTPγS-treated myc-DDK-tagged MASL1-transfected 293T cell lysate pulled-down with GST alone (negative control) (GST). (Lane 5), GDP-treated myc-DDK-tagged MASL1-transfected 293T cell lysate pull-down (inactivated-MASL1 control). (Lane 6), GTPγS-treated myc-DDK-tagged MASL1-transfected 293T cell lysate pull-down (activated-MASL1 control). (Bottom 2 panels), expression levels of MASL1 and β-actin (as an internal control) in 10% of input samples. (B) CD34⁺ cells were induced to differentiate by EPO treatment of 14 days. At day 7 (Lane 1) and 14 (Lane 2) of differentiation, protein lysates were subjected to pull-down with GST-Raf1-RBD and then western blot with anti-MASL1 antibody. (Lane 3), EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pulled-down with GST alone (negative control). (Lane 4), GDP-treated EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pull-down (inactivated-MASL1 control). (Lane 5), GTPγS-treated EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pull-down (activated-MASL1 control). (Bottom 2 panels), expression levels of MASL1 and β-actin (as an internal control) in 10% of input samples. (C) CD34⁺ cells were induced to differentiate by G-CSF (Lane 1) or EPO (Lane 2) treatment of 14 days. The protein lysates were subjected to pull-down with GST-Raf1-RBD and then western blot with anti-MASL1 antibody. (Lane 3), EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pulled-down with GST alone (negative control). (Lane 4), GDP-treated EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pull-down (inactivated-MASL1 control). (Lane 5), GTPγS-treated EPO-induced CD34⁺ cells at day 14 of differentiation protein lysate pull-down (activated-MASL1 control). The lower 2 panels show expression levels of MASL1 and β-actin (as an internal control) in 10% of input samples. Minus (−) and Plus (+) indicate the addition of GDP or GTPγS, respectively.