Western lightning ultra

Immunoblotting was performed as described previously^{39 (link)}. In short, cells were lysed and protein extracts were boiled and loaded on 10% polyacrylamide gels. After electrophoretic separation, the proteins were transferred to PVDF membranes, which were blocked with 5% milk powder in Tris-buffered saline + 0.1% Tween 20 (TBS-T) for 1 h. Incubation of primary antibody in TBS-T was performed at 4 °C overnight. Membranes were then washed and stained with secondary antibody. Chemiluminescence was elicited using Western Lightning Ultra from PerkinElmer (Waltham, MA, USA) or Clarity Western ECL Substrate from Bio-Rad Laboratories (Hercules, CA, USA), respectively, according to the manufacturers’ instructions. The following primary antibodies were used: anti-caspase 3, anti-cleaved caspase 3 (Asp175), anti-PARP, and anti-pCHK1(Ser345) (133D3) from Cell Signaling (Cambridge, UK), anti-CHK1 (G-4) and anti-POLD1 (A9) from Santa Cruz Biotechnology (Dallas, TX, USA), and peroxidase-conjugated anti-β-Actin (AC-15) from Sigma-Aldrich (Hamburg, Germany). HRP-conjugated anti-rabbit, anti-goat and anti-mouse antibodies from Santa Cruz Biotechnology were used as secondary antibodies.

3–5 × 10⁶ cells were collected per sample, resuspended in 6x Laemmli Buffer and heated at 95 °C for 10 min. The samples were subjected to SDS-PAGE gel electrophoresis using 10% polyacrylamide gels. The gels were then stained with SERVA blue G or blotted on a 0.45 µm nitrocellulose blotting membrane (Neolabs). To verify the protein transfer, the membrane was stained with Ponceau S (SERVA). The membrane was blocked with 5% milk in TBS-Tween and incubated with appropriate concentrations of first and secondary antibodies. Western Lightning Ultra (Perkin Elmer) was used as a chemiluminescence system and signals were detected with the LAS-4000 imager (GE Healthcare) and CCD camera (Fujifilm). Antibodies used were: rabbit anti-Aldolase (1:50000) (Clayton, 1987 (link)); mouse anti-myc 9E10 (Santa Cruz, 1:200); rabbit Peroxidase anti-Peroxidase (Sigma, 1:20000); rat anti-ribosomal protein S9 (1:1000); anti-Trypanothione Reductase (rabbit, gift from L. Krauth-Siegel, BZH Heidelberg); mouse anti-V5 (Biorad, 1:2000); anti-SCD6 and anti-DHH1 (from S. Kramer, University of Wurzburg, 1:10000 and 1:15000 respectively) and rabbit anti-BiP (from J. Bangs, University of Buffalo, 1:1000).

BMDCs were treated with LPS in the presence of BVDU or not for the indicated duration at 37 °C, then cells were lysed, sonicated and boiled at 95 °C for 5 min in sample buffer. BMDCs lysates were separated on SDS-PAGE on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Then the PVDF membranes were incubated with blocking buffer containing 5% non-fat milk for 1 h and later incubated overnight at 4 °C in buffer containing antibody against ERK/P-ERK (CST), JNK/p-JNK (CST), p38/p-p38 (CST), NF-κBp50, p65 (SantaCruz) or GAPDH (CST). After washing for three times, the membranes were incubated with proper HRP-conjugated secondary antibodies for 1 hour. Later after washing thrice, immunostaining was visualized using Western Lightning Ultra (PerkinElmer).