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. 2016 Nov 17;99(6):1359–1367. doi: 10.1016/j.ajhg.2016.10.006

Figure 5.

Figure 5

Knockdown of DENND5A Leads to Enhanced Erk Activation and Increased Amounts of Neurotrophin Receptors

(A) PC12 cells were transduced with control lentivirus (shRNAmiR ctrl) or lentivirus to knockdown DENND5A (DENND5A nt1758 and DENND5A nt2652). After 24 hr serum starvation, transduced PC12 cells were harvested (−NGF) or treated for an additional 24 hr with NGF (50 ng/mL) (+NGF). Equal protein aliquots of lysates prepared from these cells were processed by western blot with antibodies recognizing phospho-Erk (monoclonal antibody, Cell Signaling Technology) and total Erk (monoclonal antibody, Cell Signaling Technology). HSP90 (monoclonal antibody, Assay Designs Stressgen) was used as a loading control.

(B) Quantification of five experiments performed as in (A). The graphs show the average relative change in phospho Erk1/2 levels normalized to total Erk1/2 levels between cells with knockdown DENND5A and control cells. Error bars represent the SEM, and statistical analysis employed a one-way ANOVA followed by a Bonferroni post-test. ∗p < 0.05, ∗∗p < 0.01.

(C) After 24 hr serum starvation, transduced PC12 cells were harvested (−NGF) or treated for 24 hr with NGF (50 ng/mL) (+NGF). Equal protein aliquots of lysates prepared from these cells were processed by western blot with antibodies recognizing TrkA (polyclonal antibody for pan Trk [TrkA and TrkB] [C17F1], Cell Signaling Technology) and HSP90.

(D) Quantification of TrkA amounts from experiments as in (C) normalized to HSP90 and expressed in relation to those in control shRNAmiR cells. Data show the mean ± SEM from eight independent experiments, and statistical analysis employed a one-way ANOVA followed by a Bonferroni post-test. ∗p < 0.05, ∗∗p < 0.01.

(E) Rat cortical neurons were transduced with control lentivirus (shRNAmiR ctrl) or lentivirus to knockdown DENND5A (DENND5A nt1758 and DENND5A nt2652) at 1 DIV. Cell lysates were prepared at 8 DIV, and equal protein aliquots were processed by western blot with antibodies recognizing the indicated proteins.

(F) Quantification of TrkB levels from experiments as in (E) normalized to GAPDH and expressed in relation to those of control shRNAmiR cells. Data show the mean ± SEM from six independent experiments, and statistical analysis employed a repeated-measure one-way ANOVA followed by a Dunett’s post-test. ∗p < 0.05.