ROCK activity is involved in the regulatory effect of RES on dendrite development.

<p>Hippocampal neurons were treated at 3 DIV with 5 µM TAT-TI-JIP, 1µM SP600125 or 100 µM Y-27632 in the presence or absence of 50 µM RES. Neurons were fixed at 6 DIV and immunostained against MAP-2. (<b>A</b>) Representative images of MAP-2 immunostaining in all experimental conditions. Scale bar, 10 µm. (<b>B</b>) Quantification of TDBTN and coverage area of neurons in all conditions shown in (A). Values were normalized against control neurons. (<b>C</b>) Sholl analysis of control neurons (black circles), and neurons treated with TAT-TI-JIP (cian circle), TAT-TI-JIP + RES (red triangle), SP600125 (blue triangle), SP600125+RES (green square), RES treatment reverts the JIP and SP600125 effect significantly increasing the number of intersection at 26–50 µm from the soma compared to non treated neurons (p<0,01 at 26–50 µm). Error bars indicate S.E.M. n = 50–60 neurons. (<b>D</b>) Sholl analysis of control neurons (black circles), and neurons treated with Y−27632 (red circle), Y−27632+ RES (blue triangle). Treatment with Y−27632 in the presence or absence of RES significantly increases the number of intersection at 24–70 µm from the some compared with non treated neurons (p<0,01 at 24–70 µm). (<b>E</b>) Quantification of TDBTN in neurons treated with different doses of Y−27632 in the presence or absence of Res. (<b>F</b>) Representative immunoblot against total Myosin Phosphatase Target 1 (MYPT1) and MYPT 1 phosphorylated at T853 (pMYPT), a specific substrate of ROCK kinase. Densitometric values of pMYPT1 bands were normalized against total MYPT1, and then each condition expressed as relative to the control condition without treatment. Experiments were carried out in triplicate. Students <i>t</i>-test, *p<0.01.