Rabbit anti p38 mapk

Lipopolysaccharides (LPS) (Escherichia coli O111:B4) were purchased from Sigma Aldrich. ON-TARGETplus SMARTpool siRNA and single siRNA targeting mouse Ruvbl2 and Ruvbl1 were obtained from Dharmacon. For immunoblotting and ChIP assay, the following antibodies were used: mouse monoclonal anti-Reptin 52 (RUVBL2) (Santa Cruz), mouse anti-TIP49A (RUVBL1) (Abcam), mouse anti-β-Actin (Sigma-Aldrich), rabbit anti-p38/MAPK (Cell Signaling Technology), rabbit anti-p-p38/MAPK (T180/Y182; Cell Signaling Technology), rabbit anti-p44/42 (ERK1/2; Cell Signaling Technology), rabbit anti-p-p44/42 MAPK (ERK1/2; Thr202/Tyr204 (Cell Signaling Technology), rabbit anti-SAPK/JNK (Cell Signaling Technology), rabbit anti-p-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology), rabbit anti-IκBα (Cell Signaling Technology), rabbit anti-Stat1 (Cell Signaling Technology), rabbit anti-p-Stat1(S727) (Cell Signaling Technology), rabbit anti-p-Akt (S473) (Cell Signaling Technology), mouse anti-LAMIN B1 (Santa Cruz), mouse anti-α-tubulin (Sigma-Aldrich), rabbit anit-p50 (Abcam), rabbit anti-H3K4Me3 (Cell Signaling Technology), rabbit anti-H4K20me3 (Santa Cruz Biotechnology), and rabbit anti-p50 (Cell Signaling Technology) antibodies. CB-6644 was obtained from MedChemExpress.

P815-HTR cells were lysed in radio-immunoprecipitation assay buffer (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM Na₃VO₄, 5 mM NaF, and a protease inhibitor cocktail). A total of 30 µg of protein was separated per lane on 10% SDS-polyacrylamide gels, followed by transfer onto PVDF membranes (Millipore). The membranes were blocked in TBST (Tris-buffered saline containing 0.1% Tween-20) supplemented with 5% nonfat milk. Probing with the primary antibody was overnight at 4 °C; the blot was washed three times with TBST, and then incubated with appropriate secondary antibody (anti-rabbit HRP). After washing the blots three times in TBST, the protein bands were detected using a Western HRP substrate ECL kit (Luminata Forte, Millipore) and chemiluminescence imaging (Fusion FX, Vilber Lourmat). Primary antibodies were rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling #9211; 1: 1000), rabbit anti-p38 MAPK (Cell Signaling #9212; 1: 1000), rabbit anti-phospho-Erk1/2 (Thr202/Tyr204) (Cell signaling #9101; 1: 1000), and rabbit anti-Erk1/2 (Cell Signaling #9102; 1: 1000). Secondary antibody was goat anti-rabbit IgG HRP (Thermo Fisher; 1: 5000).

Immunoblotting was performed as in Hyvonen et al.^{54 (link)} Membranes were incubated overnight at +4 °C with rabbit anti-PDK1, rabbit anti-p38 MAPK, rabbit anti-phospho-p38 MAPK (Thr180/Tyr182), rabbit anti-phopho-Akt (Ser473), mouse anti-caspase-3 or rabbit anti-cleaved caspase-3 (Cell Signaling Technology), mouse anti-Pan Akt (R&D Systems, Minneapolis, MN, USA), rabbit anti-Bax or rabbit anti-Bcl2 (Abcam, Cambridge, UK), mouse anti-tubulin or mouse anti-actin IgGs (Sigma-Aldrich), followed by Alexa Fluor 680 (Invitrogen) or IRDye 800 (LI-COR, Lincoln, NE, USA) donkey anti-rabbit, anti-goat or anti-mouse IgGs. Detection and quantification was performed with an Odyssey Infrared Imager (LI-COR).

For immunocytochemistry, samples (neurospheres or neurons: derived from the 201B7 line) were plated onto poly-L-ornithine/fibronectin-coated chamber slide glasses (Iwaki) and fixed in 4% PFA/PBS for 30 min at room temperature. The slides were rinsed with PBS three times and permeabilized with 0.3% Triton X-100/PBS for 5 min at room temperature. After blocking with Blocking One (Nacalai Tesque, 03953-95) for 15 min at room temperature, the slides were incubated at 4°C overnight with the following antibodies: rabbit anti-α-tubulin (Cell Signaling Technology, 2144; 1:500), mouse anti-β-III tubulin (Sigma-Aldrich, T8660-2ML; 1:500), rabbit anti-tau (Dako, A0024; 1:500), rabbit anti-p38 MAPK (Cell Signaling Technology, 8690; 1:500), rabbit anti-phospho-p38 MAPK (Cell Signaling Technology, 4511; 1:500), rabbit anti-phospho-CDC25B (Thermo Fisher Scientific, PA5-104568; 1:500), and rabbit anti-acetyl-α-tubulin (Lys40) (Cell Signaling Technology, 5335; 1:500). After washing three times with PBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 (Thermo Fisher Scientific, A-11034) or Alexa 555 (Thermo Fisher Scientific, A-21424; 1:500) for 60 min at room temperature and then subjected to nuclear counterstaining with Hoechst 33258 (Sigma-Aldrich, B2883; 10 μg/mL). The samples were analyzed with an all-in-one fluorescence microscope (BZ-700 or BZ-800, Keyence).

Lipoteichoic acid (LTA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38MAPK inhibitor SB203580 and NF-κB inhibitor Bay11-7082 were purchased from Calbiochem (San Diego, CA, USA). The rabbit anti-p38MAPK, rabbit anti-phospho-p38MAPK, rabbit anti-IκBα, rabbit anti-phospho-IκBα, rabbit anti-NF-κB p65 antibodies, and normal rabbit IgG were purchased from Cell Signaling (Beverly, MA, USA). The mouse anti-ACTB and rabbit NF-κB p65 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The rabbit anti-TLR2 and rabbit anti-DEFB131 antibodies were purchased from Abcam (Cambridge, UK).

Proteins obtained as described previously^{58 (link)} were blotted onto polyvinylidene fluoride membrane (Calbiochem/Merck Millipore) and incubated with following antibodies: rabbit anti-NTSR-1 (ANT-015), rabbit anti-NTSR-2 (ANT-016), Alomone Labs; rabbit anti-Bcl-2 (sc-783, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-Bcl-xL (#2764S), rabbit anti-phospho-Akt (Ser473) (#4060S), mouse anti-Akt (pan; #2920S), rabbit anti-phospho-Src family (Tyr416; #6943S), rabbit anti-Src (#2108S), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182; #9211S), rabbit anti-p38 MAPK (#8690S), rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185; #4668S), and rabbit anti-SAPK/JNK (#9252), all from Cell Signaling Technology (Danvers, MA, USA); mouse anti-Giα1/2 antibody (06-236, Calbiochem/Merck Millipore); and anti-actin (A5441, Sigma-Aldrich). After washing (tris-buffered saline/0.1% Tween-20), the immunoreactions were detected by incubation for 2 h at RT with horseradish peroxidase-conjugated secondary Ab against mouse or rabbit Ig (P0447 and P0448, respectively, Agilent Technologies, Santa Clara, CA, USA), revealed with the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). Western blot were detected using Bioimaging Systems (GeneSnap and GeneTool; Syngene, Cambridge, UK). Densitometric analyses were performed using the ImageJ software (NIH, Bethesda, MD, USA).