Anti phospho jnk

Spautin-1(S7888), 3-methyladanine (3-MA, S2767), Z-VAD-FMK (S7023), SP600125 (S1460), SB230580 (S1076), LY3214996 (S8534), Gefitinib (S1025) and Enzalutamide (MDV3100, S1250) were purchased from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was obtained from Xcessbio Biosciences, Inc. (San Diego, CA). USP10 (sc-76,811), USP13 (sc-76,815) and Glut1 (sc-35,493) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies: anti-Glut1 (ab652), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630), anti-Ki67 (BS1454) (Bioworld Technology, Inc., Louis Park, MN, USA); anti-USP10(#8501), anti-SKP2(#2652), anti-p27(#3686), anti-CDK4(#12790), anti-CDK2(#2546), anti-CyclinD1(#2978), anti-p15(#4822), anti-p21(#2947), anti-PARP(#9532), anti-Bim(#2933), anti-Bax(#14796), anti-Bcl-2(#15071), anti-activated Caspase-3(#9664), anti-phospho-JNK (#9255), anti-JNK(#9252), anti-phospho-ERK1/2(#4370), anti-ERK1/2(#4695), anti-phospho-p38(#4511), anti-p38(#8690), anti-phospho-MKK4(#4514), anti-MKK4(#9152), anti-phospho-MEK1/2 (#9154), anti-MEK1/2(#4694), anti-phospho-EGFR (Tyr1173)(#4407), anti-phospho-EGFR (Tyr1068)(#3777), anti-EGFR(#2085) (Cell Signaling Technology, Beverly, MA, USA).

RT–QPCR(Realtime-Quantitative reverse transcription polymerase chain reaction) and Western blot were done as previously described [33 (link)]. RT-QPCR primers were designed using Primer 5.0 software. Sequences of the primers are provided in Additional fie 1: Table S3.
Western blot was performed according to standard methods as described previously [34 (link)]. Anti-Siah1 (Abcam, #ab69638, dilutions: 1:200), anti-phospho-AKT (Cell Signaling Technology, #C31E5E, dilutions: 1:200), anti-AKT (Cell Signaling Technology, #11E7, dilutions: 1:1000), anti-JNK (Bioworld Technology Inc., #BS1544, dilutions: 1:200), anti-phospho-JNK (Bioworld Technology Inc., #BS4322, dilutions: 1:1000), and anti-YAP (Cell Signaling Technology, #8418, dilutions: 1:200) antibodies were used for Western blot. A mouse monoclonal anti-α-Tubulin antibody (Tianjin Sungene Biotech Co, #KM9007, dilutions: 1:10,000) was used as an internal control to confirm equal loading of proteins.

Escherichia coli 0127:B8 was obtained from ATCC (12740). LPS (Escherichia coli 0127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rolipram was obtained from Enzo Life Science (Farmingdale, NY, USA). Rabbit anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were procured from Bioworld Technology (St. Louis, MN, USA). Rabbit anti-NFκB p65 and anti-phospho-NFκB p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor® 488 goat anti-rabbit IgG (H + L) was purchased from Molecular Probes (Eugene, OR, USA). MILLIPLEX® MAP Mouse Cytokine/Chemokine Magnetic Bead Panel Kit for 96-Well Plate Assay was purchased from EMD Millipore (Darmstadt, Germany).