Anti phospho p42 44

Following drug treatment, larvae were snap-frozen in liquid nitrogen and protein was extracted using lysis buffer: 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 30 mM NaF, with PhosStop and CompleteMini inhibitors (Roche). Larvae were disrupted using a hand-held pestle and allowed to incubate on ice for 15 min, prior to spinning at 14,000 rpm for 15 min at 4°C. Protein samples were run on gradient gels (4%–20% TGX-gels; Bio-Rad), and transferred to an Immobilon-FL PVDF membrane (Millipore). Blocking and antibody incubation was done in Odyssey Blocking Buffer diluted 1:1 in PBS. Antibodies were anti-phospho-p42/44 (1:2,000; Cell Signaling Technology) and anti-p42/44 (1:1,000; Cell Signaling Technology). Secondary antibodies were IRDye labeled donkey anti-mouse antibody (1:15,000) and goat anti-rabbit antibody (1:15,000).

For Western blot analyses, cells were plated on 6-well plates and were serum-starved overnight. Cells were treated with compounds accordingly, and protein samples were prepared as described previously [36 (link)]. Then, 30 µg of total proteins was loaded to each well, and proteins were separated using 4–12% Tris Glycine Precast Gel (KOMA BIOTECH, Seoul, Republic of Korea). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Blocking was carried out using Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% BSA at room temperature for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4 °C with the indicated antibodies: anti-p42/44 (Cell Signaling; Cat#9102S) and anti-phospho-p42/44 (Cell Signaling; Cat#9101L). Subsequently, the membranes were washed out with TBST 3 times at 5 min intervals and incubated with HRP-conjugated anti-secondaries for 1 h at room temperature. Finally, visualization was carried out using an ECL Plus immunoblotting detection system (GE Healthcare, Piscataway, NJ, USA). All experiments were repeated five times independently, and ImageJ software (NIH, Bethesda, MD, USA) was used for analysis.

M-CSF MФ and GM-CSF MФ were infected with mock or LCMV for 1 h, and then stimulated with R848 for 15 min. Cells were then harvested and pellets were incubated in lysis buffer (1 M HEPES, 0.5 M NaF, 0.5 M EGTA, 2.5 M NaCl, 1 M MgCl₂, 10 % glyercol, 1 % Triton X-100) with PhosSTOP phosphatase inhibitor (Roche, Switzerland). The Bradford assay (BioRad Laboratories, USA) was used to determine protein concentration. Lysates were subjected to electrophoresis on 10 % polyacrylamide SDS-PAGE and transferred to polyvinylidene difluoride membrane (BioRad Laboratories). Membranes were probed with the following primary antibodies: rabbit anti-phospho-NF-κB (Santa Cruz Biotechnology, USA), anti-phospho-p42/44, anti-phospho-p38 (Cell Signaling Technologies, USA) and secondary antibody : goat anti-rabbit-HRP (Santa Cruz Biotechnology). Membranes were stripped and reprobed with rabbit anti-NF-κBp65, anti-pan p38, or anti-p42/44 (Santa Cruz Biotechnology). All membranes were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and imaged and quantified using an Alpha Innotech FluorChem HD2 system using AlphaView software version 3.1.0.0. To obtain fold change data, the densitometry of phospho-specific bands was first normalized to respective pan bands and the mock medium control for each of M-CSF and GM-CSF cells was used to calculate the fold change.