Skip to main content
. 2014 Sep 1;33(21):2447–2457. doi: 10.15252/embj.201489385

Figure 4.

Figure 4

SOCS6 regulates YAP ubiquitylation

  1. Co-immunoprecipitation of SOCS6 with YAP1. HEK293T cells were transfected to express HA-tagged SOCS6 and treated with MG132 to block proteasome activity. Anti-YAP1 antibody was used for IP. Blots were probed with anti-HA to visualize HA-SOCS6. Total YAP1 was lower in cells expressing SOCS6. Co-transfection with ubiquitin increased recovery of SOCS6 in the YAP1 IP (not shown). 30% of the IP was loaded for immunoblot analysis. Lysate: 5% of the input.
  2. Effect of depleting SOCS5 and SOCS6 on YAP1 ubiquitylation. BJ cells were transfected with siRNAs to target SOCS5, SOCS6 or with a control siRNA. Cells were co-transfected with ubiquitin and treated with MG132 (+) or DMSO control (−) for 5 h before IP with anti-YAP1. Upper panel probed with anti-ubiquitin (Ubi). Lower panel probed with anti-YAP1. All samples were run on the same gel with an intervening lane removed (space between lanes 2 and 3).
  3. Effect of SOCS5 and SOCS6 overexpression on YAP1 ubiquitylation. Samples prepared as in (D).
  4. Co-IP of SOCS6 with phosphosite mutant YAP1. HEK293T cells were transfected to express HA-tagged SOCS6 and Flag-tagged YAP1 or YAP1S127A/S397A. Cells were treated with MG132. Anti-Flag antibody was used for IP. Blots were probed with anti-Flag to visualize recovered Flag YAP1 proteins and with anti-HA to visualize HA-SOCS6. All samples were run on the same gel with an intervening lane removed. 30% of the IP was loaded for immunoblot analysis.
  5. Effect of SOCS6 overexpression on YAP1 and YAP1S127A/S397A ubiquitylation. Samples prepared as in (D).
  6. Effect of SOCS6 overexpression on colony formation in BJT/p53/p16KD/ST cells expressing YAP1 or YAP1S127A/S397A. Colony number is the average of three independent experiment normalized to YAP1 control cells without SOCS6 overexpression ± SD. P < 0.05 comparing SOCS6-expressing cells to their respective controls.
  7. HEK293T cells were transfected with Flag-tagged YAP1, HA-tagged SOCS6 and with siRNAs targeting Elongin B/C or Cullin-5. Left panel: samples were immunoprecipitated with anti-Flag and probed with the indicated antibodies. SOCS6 overexpression increased ubiquitylation of Flag-tagged YAP1. This increase was limited by depletion of either Elongin B/C or Cullin-5. Endogenous SOCS6 was recovered at lower level in the Flag-IP sample without SOCS6 overexpression.

Source data are available online for this figure.