α tubulin

Rat brain area homogenates (100μg) were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA). The blots were blocked in 2% (w/v) non-fat dried milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween-20) and incubated with α-GBA (Sigma, St. Louis, USA), α-GBA2 (Eurogentec custom made) and α-Tubulin (Cedarlane Laboratories, Ontario, Canada) primary antibodies diluted in blocking buffer, overnight at 4°C. Blots were washed for 30 minutes in TBST. After washing, the membranes were incubated with matching IRDye-conjugated secondary antibodies (Westburg BV, Leusden, Netherlands) for 1 hour at room temperature. Blots were scanned on an Odyssey image scanner (GE Healthcare).

For western blotting, cells were resuspended in lysis buffer containing protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche). Gel elecrophoresis was performed using 10% polyacrylamide gels, and blotting was performed using the Trans-blot Turbo Transfer System (Bio-rad, The Netherlands) and the Trans-blot turbo RTA Transfer kit (LF PVDF, Bio-rad). Antibodies used were the following: ERK1/2 (137F5; 4695; Cell Signaling, The Netherlands), P-ERK1/2 (E-4;SC-7383; Santa Cruz Biotechnology, Texas), JNK1/2 (9252; Cell Signaling), P-JNK1/2 (81E11; 4668; Cell Signaling), P38 (9212; Cell Signaling), P-P38 (9211; Cell Signaling), P-STAT1 (Tyr-701; 9167; Cell Signaling), α-Tubulin (Cedarlane, Canada) and PARP1 (4C10-5, BD Biosciences, The Netherlands). As secondary antibodies we used Goat-anti Rabbit IRDye 800CW/680RD (LI-COR Biosciences, Nebraska), Goat-anti-mouse IRDye 800CW/680RD (LI-COR Biosciences). Imaging was performed on the Odyssey (LI-COR Biosciences), and quantification of signal intensity was done using Image Studio (LI-COR Biosciences). The signal intensity of phosphoproteins was corrected for the signal intensity of the (total) unphosphorylated protein (ERK/P38/JNK) or for the signal intensity of a housekeeping gene (α-Tubulin to correct for STAT1α/β).

THP-1 cells, in 12-well plates, were stimulated by 1.0 μg ml⁻¹ poly(dA:dT) (Invivogen) coated with Lipofectamine 2000 (Invitrogen) for 2 h. For the qPCR, cell lysis and reverse-transcription were performed with a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo), according to the manufacturer’s instructions. Quantitative real-time PCR was performed with Power SYBR Green PCR Master Mix (Life Technologies), and a Step-One-Plus PCR system (Applied Biosystems) by the ΔΔCT method, using the following oligonucleotides: IFNB1 sense, 5′-AGGACAGGATGAACTTTGAC-3′, and IFNB1 antisense, 5′-TGATAGACATTAGCCAGGAG-3′; IFIT2 sense, 5′-TGGTGGCAGAAGAGGAAGAT-3′, and IFIT2 antisense, 5′-GTAGGCTGCTCTCCAAGGAA-3′; and GAPDH sense, 5′-AGCAACAGGGTGGTGGAC-3′, and GAPDH antisense, 5′-GTGTGGTGGGGGACTGAG-3′. For the immunoblotting experiments, the cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100) containing complete protease inhibitor (Roche), and immunoblotting was performed using the following antibodies: DDX41 (H00051428-M01, Abnova), P-IRF3 (#4947, Cell Signaling Technology), IRF3 (#11904, Cell Signaling Technology), and α-tubulin (Cedarlane).