Journal: The EMBO Journal
Article Title: RASSF 1A is required for the maintenance of nuclear actin levels
doi: 10.15252/embj.2018101168
Figure Lengend Snippet: Co‐immunoprecipitation of endogenous RASSF1A with endogenous RAN, CRM1/XPO1, XPO4, XPO5, XPO6 and XPO7 from HeLa cell lysates, compared with the IgG control. Upper: graphical representation of the domain structure of full‐length RASSF1A and mutant constructs used for mapping RASSF1A/XPO6 interaction. Different domains are abbreviated as follows: SARAH, Salvador‐RASSF‐Hippo domain; C1, N‐terminal C1 type zinc fingers; and RA, Ras‐binding domain. Lower: Western blot analysis of MYC immunoprecipitation from the indicated inputs from HeLa cells. Co‐immunoprecipitation of endogenous XPO6 with RAN in siRASSF1A from HeLa cell lysates. Co‐immunoprecipitation of endogenous XPO6 with endogenous RAN, RASSF1A and MST2 in siRASSF1A and siMST2 HeLa cells. Quantification of the interaction of XPO6 with RASSF1A, RAN and MST2 relative to XPO6 is shown. Error bars derive from three independent experiments and represent the SEM. Co‐immunoprecipitation of endogenous RASSF1A with endogenous XPO6 and RAN in siRNA‐mediated knockdown of MST2 HeLa cells. Quantification of the interaction of RASSF1A with XPO6, RAN and MST2 relative to RASSF1A is shown. Error bars derive from three independent experiments and represent the SEM. Data information: Two‐tailed Student's t ‐test was used for statistical analysis. * P < 0.05, ** P < 0.01. Source data are available online for this figure.
Article Snippet: The following antibodies were used in this study: RASSF1A (Atlas, HPA040735), MST1 (Sigma, WH0006789M1), MST2 (Abcam, ab71960, ab52641), XPO6 (Bethyl, A301‐205A), CRM1 (Cell Signaling, 46249), XPO4 (Novus Biologicals, NB100‐56495), XPO5 (sc‐166789), XPO7 (sc‐390025), Lamin A/C (Cell Signaling, 4777), Lamin B (Abcam, ab16048), Profilin (Novus Biologicals, NBP2‐02577), IPO9 (Abcam, ab52605), Myc‐Tag (JBW301, Millipore, 05‐724), GAPDH (Cell Signaling, 97166), Actin (sc‐1616, Santa Cruz), RAN (Santa Cruz Biotechnology, sc58467), MRTF‐A (Santa Cruz Biotechnology, sc‐398675), α‐tubulin (Abcam, ab7291) and pS131‐RASSF1A custom‐made (Hamilton et al , ).
Techniques: Immunoprecipitation, Control, Mutagenesis, Construct, Zinc-Fingers, Binding Assay, Western Blot, Knockdown, Two Tailed Test