Fig. 7

Figure 7. Disturbed c-Kit degradation and trafficking affect downstream targets in Clasp2−/− mouse ESCs (A) WBs for c-Kit and tubulin (loading control) on lysates of Clasp2+/+ and Clasp2−/− mouse embryonic stem cells (mESCs) cultured overnight alone or with BafA or SCF. (B) WBs for c-Kit and tubulin (loading control) on lysates of Clasp2+/+ and Clasp2−/− mESCs cultured overnight with SCF or SCF followed by BafA. (A and B) The c-Kit band intensities were quantified using tubulin as a control; these normalized intensities are indicated in blue below gel images, with the left lanes (Clasp2+/+ culture [A] or Clasp2+/+ culture with SCF [B]) set at 100 and the other intensities calculated relative to these values. (C) Mouse Clasp2+/+ and Clasp2−/− ESC lysates were treated with EndoH or PNGase F and examined by WB using antibodies against the indicated proteins (tubulin, loading control). Shown is 1 representative blot of 2. (D) Clasp2+/+ and Clasp2−/− mESCs were treated overnight with tunicamycin, and lysates were examined by WB using antibodies against the indicated proteins (tubulin, loading control). M, mature c-Kit; N, newly synthesized c-Kit. Shown is 1 representative blot of 2. (E) Ratio of mature c-KIT levels versus newly synthesized c-KIT, measured by quantifying M and N c-Kit band intensities on WB in Clasp2+/+ and Clasp2−/− mESC lysates in 2 independent experiments (Exp #1 [shown in D] and #2). (F) WB containing Clasp2+/+ and Clasp2−/− mESC lysate were probed with antibodies against total Akt and phospho-Akt (P-Akt) and against tubulin, which served as a loading control. Shown is 1 representative blot of 4, with two independent sets of biological replicates. (G) Graph representing the relative levels of phospho-Akt to total Akt after normalization to the tubulin loading control. (H) Graph representing the relative mRNA levels of STAT3 in Clasp2+/+ and Clasp2 −/− mESCs. qRT-PCR was performed using STAT3 primers and Gapdh, used as a housekeeping control. n = 3 with 3 biological replicates per condition. ∗∗p < 0.01, ∗p < 0.05, Mann-Whitney U test, mean ± SEM (G and H).