Rabbit anti sapk jnk

BMDMs were treated as indicated. Whole-cell lysates were prepared in RIPA buffer and subjected to western blotting. The antibodies used were rabbit anti-HP1-α, rabbit anti-H3K4me3, rabbit anti-H3K9me3, rabbit anti-H3K27me3, rabbit anti-H3K27Ac, rabbit anti-H, rabbit anti-NF-B, rabbit anti-ERK, rabbit anti-MAPK, rabbit anti-p-SAPK-JNK, rabbit anti-NF-κB, rabbit anti-ERK, rabbit anti-MAPK, rabbit anti-SAPK-JNK, mouse anti-actin, HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated sheep anti-mouse IgG (all purchased from Cell Signaling Technology, Danvers, MA, USA). The signals were detected by chemiluminescence. The protein bands intensities were quantitated using ImageJ Gel Analysis program. The modified proteins were normalized to their loading controls (total forms) and relatively normalized to those of the unstimulated cells.

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).

HeLa, HEK293T, and RAW 264.7 cells were obtained from American Type Culture Collection, and MEF cells were a gift from A. Hoffmann (University of California Los Angeles). Glutathione–agarose beads and Ni–nitrilotriacetic acid (NTA) agarose beads were purchased from BioBharati LifeScience Pvt Ltd. Mouse anti-HA antibody was purchased from BioLegend. Rabbit anti-NEMO, rabbit anti-IKK2, rabbit anti-His, rabbit anti-Ub, rabbit anti-Myc, rabbit anti-γ-actin, and rabbit anti-ERK2 were purchased from BioBharati LifeScience Pvt Ltd. Mouse anti-p84 was purchased from GeneTex. Mouse anti-GST, rabbit anti-p65/RelA, and rabbit anti-IκBα were from Santa Cruz Biotechnology. Rabbit anti-p100/p52 was a gift from N. Rice (National Cancer Institute; Frederick, MD). Rabbit anti–phospho-SAPK (stress-activated protein kinase)/JNK (Thr183/Tyr185), rabbit anti-SAPK/JNK, rabbit anti–phospho-p38 MAPK (Thr180/Tyr182), rabbit anti-p38 MAPK, rabbit anti–phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and rabbit anti–phospho-IKKα/β (Ser177/181) were purchased from Cell Signaling. Horseradish peroxidase–conjugated anti-rabbit and antimouse secondary antibodies were from BioBharati LifeScience Pvt Ltd as was mouse TNF-α. LPS was purchased from Sigma. Mouse LTβR was purchased from Abcam.

Western blot procedures were performed as previously described [16 ]. The primary antibodies were rabbit anti-calcium/calmodulin-dependent protein kinase II (CaMKII) (1 : 1000, ab52476, Abcam, Cambridge, United Kingdom), rabbit anti-phospho- (P-) CaMKII (1 : 1000, ab5683, Abcam), rabbit anti-AKT (1 : 1000, #4685, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P-AKT (1 : 1000, #4060, Cell Signaling Technology), rabbit anti-P-ERK1/2 (1 : 1000, #4370, Cell Signaling Technology), rabbit anti-ERK1/2 (1 : 1000, #4695, Cell Signaling Technology), rabbit anti-P-p38 (1 : 1000, #9215, Cell Signaling Technology), rabbit anti-p38 (1 : 1000, #9212, Cell Signaling Technology), rabbit anti-P-SAPK/JNK (1 : 1000, #4668, Cell Signaling Technology), rabbit anti-SAPK/JNK (1 : 1000, #9258, Cell Signaling Technology), rabbit anti-TLR4 (1 : 1000, #14358, Cell Signaling Technology), rabbit anti-NF-κB p65 (1 : 1000, #8242, Cell Signaling Technology), rabbit anti-P-NF-κB p65 (1 : 1000, #3033, Cell Signaling Technology), and rabbit anti-β-actin (1 : 2000, #4970, Cell Signaling Technology). Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (1 : 5000, A8275, Sigma-Aldrich) was used as a secondary antibody.

Cell lysates were homogenized in lyses buffer (Beyotime, China) and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The analysis of protein was performed according to standard SDS–PAGE. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) plus detection reagent (Pierce, Rockford, IL) and analyzed using an Omega 16ic Chemiluminescence Imaging System (Ultra-Lum, CA). The following primary antibodies were used: rabbit anti-Phospho-p38 MAPK (4511, Cell Signaling Technology, USA), rabbit anti-p38 MAPK (8690, Cell Signaling Technology, USA), rabbit anti-Phospho-p44/42 MAPK (4370, Cell Signaling Technology, USA), rabbit anti-p44/42 MAPK (4695, Cell Signaling Technology, USA), rabbit anti-Phospho-SAPK/JNK (4668, Cell Signaling Technology, USA), rabbit anti-SAPK/JNK (9252, Cell Signaling Technology, USA), rabbit anti-Phospho-IKKα/β (2697, Cell Signaling Technology, USA), rabbit anti-IKKβ (8943, Cell Signaling Technology, USA), rabbit anti-Phospho-NF-κB p65 (3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (8242, Cell Signaling Technology, USA).

BMMs were activated as indicated, and the protein lysates were subjected to Western blots. The primary antibodies used in this study were as follows: rabbit anti-Notch1 (1:2000) (Santa Cruz Biotechnology, USA), rabbit anti-cleaved Notch1 (Val1744) (1:1000), rabbit anti-phospho-p38 (1:2000), rabbit anti p38 (1:2000), rabbit anti-phospho-p44-42 (1:4000), rabbit anti p44-42 (1:4000), rabbit anti-phospho-SAPK-JNK (1:2000), rabbit anti-SAPK-JNK (1:2000), rabbit anti-phospho-AKT (1:2000), rabbit anti-AKT (1:2000) and rabbit anti-RBPJSHU (1:1000) (all from Cell Signaling Technology, USA), mouse anti β-actin (1:1000) (Chemicon-Millipore, USA) and rabbit anti-GAPDH (1:4000) (Santa Cruz Biotechnology, USA). The secondary reagents conjugated with horse-radish peroxidase (HRP) were as follows: donkey anti-rabbit IgG (1:2000–1:4000) and sheep anti-mouse IgG (1:5000) (Amersham Biosciences, UK). The signals were detected by chemiluminescence on X-ray films.

The following antibodies were used: mouse anti-GAPDH [6C5], rabbit anti-lamin B1, rabbit anti-cytochrome c [EPR1327], and rabbit anti- carbamoyl phosphate synthetase-1 (CPS1) [EPR7493] (Abcam, Cambridge, UK); mouse anti-β-tubulin; mouse anti-pan actin, mouse anti-hsp60 clone LK2 and mouse anti-Bax (Thermo Scientific, Waltham, MA); and rabbit anti-SAPK/JNK (Cell Signaling, Danvers, MA).