Rabbit anti phospho jnk thr183 tyr185

Immunostaining of flat-prepped stage 16 Drosophila embryos was performed using the following primary antibodies: mouse anti-Fas 2 (1:100, clone 1D4, DHSB), mouse anti-axons CNS (1:100, BP 102, DHSB), mouse anti-Futsch (1:1000, 22c10, DHSB), rabbit anti-phospho-JNK (Thr¹⁸³ / Tyr¹⁸⁵) (1:100, Cell Signaling #9251), rabbit anti-Dcp-1 (Asp216) (1:100, Cell Signaling #9578), rabbit anti-GFP tag polyclonal (1:600, Thermo Fisher Scientific), rat anti-Elav (1:1000, clone 7E8A10, DHSB), mouse anti-Repo (1:100, clone 8D12 DHSB), mouse anti-Even-skipped (1:100, clone 3C10, DHSB), mouse anti-Engrailed (1:100, clone 4D9, DHSB) and TRITC-conjugated goat anti-HRP (1:200, Jackson ImmunoResearch #123-025-021).
The secondary antibodies used for detection were: Goat anti-Rabbit IgG (H + L), Alexa Fluor 488 conjugate (A-11008), Goat anti-Rabbit IgG (H + L) Alexa Fluor 555 conjugate (A-21428), Goat anti-Mouse IgG (H + L) Alexa Fluor 488 conjugate (A-11001), Goat anti-Mouse IgG (H + L) Alexa Fluor 555 conjugate (A-21422) and Goat anti-Rat IgG (H + L) Alexa Fluor 555 conjugate (A-21434). All secondary antibodies were used in a dilution of 1:600 and were from Invitrogen.

Etoposide (Sigma, St. Louis, MO, USA) was dissolved in DMSO and added at the final concentration 5 µM for 1 h. Whole cell lysates were prepared from freshly collected cells by using a lysis buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 0,5% Nonidet P-40, 400 mM NaCl) supplemented with protease inhibitor mixture (Calbiochem, Merck Darmstadt, Germany), 0.5 mM PMSF and 2 mM sodium orthovanadate. Lysates were incubated on ice for 15 minutes and centrifuged at 13000 rpm for 10 min. Protein concentration from supernatants was determined by Biorad assay (Bio-Rad Laboratories, CA, USA). For immunoblots, samples were loaded in Laemmli buffer on 6% or 10% Tris-glycine SDS/PAGE gels, transferred to nitrocellulose membranes and hybridized with appropriate antibodies at 1∶1000 dilution. Blots were developed by enhanced chemiluminescence (Roche Diagnostic GmbH, Mannheim, Germany). Antibodies: mouse anti-phosphoAtm (Ser 1981) rabbit anti-phosphoChk1 (Ser 345), rabbit anti-phosphoChk2 (Thr 387), rabbit anti-phospho JNK (Thr183/Tyr185) (Cell Signaling, New York, NY, USA), rabbit anti -p53 and mouse anti-MCM7 (Santa Cruz).

Protein content was quantified using the Bradford method. The samples were resolved by 10%–15% SDS-PAGE and transferred onto PVDF membranes (Millipore). After that, the membrane was blocked with 5% fat-free milk or BSA for 20–30 min and washed with TBST, followed by incubation with the indicated primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-JNK (Cell Signaling Technology, 9252), rabbit anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling Technology, 4668), rabbit anti-p38 (Cell Signaling Technology, 2308), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, 4511), rabbit anti-ATF2 (Cell Signaling Technology, 9226s), rabbit anti-phospho-ATF2 (Cell Signaling Technology, 5112), mouse anti-β-actin (Santa Cruz, SC-47778), rabbit anti-H2AX (Cell Signaling Technology, 7631), rabbit anti-phospho-Histone H2AX (Ser139) (Cell Signaling Technology, 3377s), and mouse anti-GST-tag (Proteintech Group, 66031-1-Ig). The chemiluminescence method (Bio-Rad) was used to detect the signals.