Anti jip1

Total cellular proteins were prepared in RIPA lysis buffer with phosphatase inhibitor cocktail and protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Twenty micrograms of total proteins were loaded into 12% Tris-acrylamide gels. The antibodies used in our studies were anti-β-actin (Santa Cruz, sc-47778), anti-phospho-JNK (Thr183/Tyr185) (Wanlei, H01291813), anti-JNK (Santa Cruz, sc-571), anti-β-catenin (Cell Signaling Technology, #8480), anti-P53 (Santa Cruz, sc-126), anti-HPV18 E6 (Santa Cruz, sc-365089), anti-WNT4 (Santa Cruz, sc-376279), anti-JIP1 (Abcam, ab24449), anti-JIP2 (Santa Cruz, sc-53553), and anti-STMN3 (Abcam, ab171625).

Immunofluorescence experiments were performed according to [73 (link)]. Briefly, hippocampal primary cultures at DIV14 (see above) were fixed in 4% paraformaldehyde (PFA) and 4% sucrose solution for 30 min, followed by permeabilization with phosphate-buffered saline (PBS) at pH 7.4, containing 0.5% Triton X-100, for 3 min. Co-cultures were first blocked for 1 h in PBS containing 1% BSA, 0.2% Triton X-100, and subsequently incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100. The following antibodies were used: anti-GFP (AbCam Cambridge, UK Ab290), anti-PSD95 (AbCam Cambridge, UK Ab12093), anti-JIP1 (AbCam Cambridge, UK Ab24449), anti-β-arrestin2 (AbCam Cambridge, UK Ab31294), anti-JNK3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA #PA5-14421). Cells were finally incubated with secondary antibodies (AlexFluor Antibody, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. A concentration of 2 mg/mL Hoechst (Thermo Fisher Scientific, Waltham, MA, USA, 33342) was used to stain nuclei. ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) was used as a mounting agent.