Polyclonal goat anti rabbit immunoglobulin hrp

Proteins were mixed with 5 × Laemmli buffer and incubated at 100 °C for 10 minutes. Samples were immediately transferred in the ice afterwards. All samples and prestained protein ladder (10-250 kDa, Thermo ScientificTM) were loaded into the columns of NuPAGETM 4-12% Bis-Tris Gel (Invitrogen) placed in the Invitrogen tank containing 1× NuPAGE MOPS SDS running buffer (Novex). The gel was run at 100v for 75 minutes and transferred to a PVDF (Polyvinylidene difluoride) membrane (Millipore) using NuPAGE transfer buffer (Novex) with methanol and antioxidant (Invitrogen) at 35v for 60 minutes. The membrane was blocked with 5% milk-TBST at RT for 1 hour and incubated overnight with TRIB1 (Millipore), and HSP90 (Abcam) diluted in 5% milk-TBST (1:1000 and 1:5000 respectively) at 4 °C. The membrane was then washed with 0.1 v/v TBST for 5 minutes 3 times and incubated with Polyclonal Goat anti-Rabbit Immunoglobulin/HRP, and Polyclonal Rabbit anti-Rat Immunoglobulin/HRP (Dako) diluted in 5% milk-TBST (1:2500 and 1:5000 respectively) at RT for 1 hour. The membrane was then washed with TBST 3 times for 5 minutes, incubated with ECL, and imaged with Bio-Rad imager.

Radioactive arachidonoyl ethanolamide[1-³H] ([³H]-AEA) was obtained from American Radiolabeled Chemicals, Inc (St Louis, MO, USA). (R)(-)-Flurbiprofen and (S)(+)-Flurbiprofen were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Ovine COX-1 (cat. no. 60100), human recombinant COX-2 (cat. no. 60122), COX-2 polyclonal antibody (rabbit anti-mouse, cat #: 160106), arachidonic acid, 2-AG, AEA and URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) were purchased from the Cayman Chemical Co. (Ann Arbor, MI, USA). Substrates were dissolved in ethanol or DMSO as appropriate. Polyclonal goat anti-rabbit immunoglobulin/HRP was obtained from Dako (Glostrup, Denmark). Protease inhibitor cocktail set III was obtained from Merck Millipore (Darmstadt, Germany). For the lipid quantification experiments, the following native and deuterated standards were purchased from the Cayman Chemical Co.: AEA, 2-AG, PEA, OEA, DEA, LEA, SEA, 2-LG, AEA-d⁸, 2-AG-d⁸, PEA-d⁴, SEA-d³, OEA-d⁴, PGF_2α, PGE₂, TXB₂, PGD₂, 12(13)-EpOME, 9(10)-DiHOME, 12(13)-DiHOME, 11-HETE, 12-HETE, 15-HETE, 9-HODE, 13-HODE, 12-HEPE, 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA), 12(13)-DiHOME-d⁴, 12(13)-EpOME-d⁴, 9-HODE-d⁴, PGE₂-d⁴ and PGD₂-d⁴. 9,10,13-TriHOME and 9,12,13-TriHOME were obtained from Larodan (Sweden, Malmö). For list of lipid abbreviations, see S1 Table.

Total cell protein extraction and immunoblot analysis was performed as previously described.^{65 (link)} For the extraction of total protein from UVB-irradiated cells, culture media were also collected by centrifugation to include any dead or apoptotic cells in the medium. Primary antibodies used were: goat polyclonal anti-14-3-3σ (N-14, sc-7681, Santa Cruz, Heidelberg, Germany), mouse monoclonal anti-Bcl-2 (100, sc-509, Santa Cruz), rabbit polyclonal anti-Caspase-3 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-EGFP (ab-290, Abcam, Cambridge, UK), rabbit polyclonal anti-GLI2 (H300, Santa Cruz), mouse monoclonal anti-p21^WAF1/CIP1 (Santa Cruz), mouse monoclonal anti-p53 (DO1, CRUK, Lincoln's Inn Fields, London, UK) and mouse monoclonal anti-β-actin, Sigma-Aldrich). Secondary HRP antibodies were as follows: polyclonal rabbit anti-mouse immunoglobulin/HRP (DakoCytomation, Cambridgeshire, UK), polyclonal goat anti-rabbit immunoglobulin/HRP (DakoCytomation) and polyclonal rabbit anti-goat immunoglobulin/HRP (DakoCytomation).