Anti myc

The anti-PHF14 (against GST-fused PHF14 1-160 a.a.), anti-KIF4A (against GST-fused KIF4A 858-1232a.a), and anti-GST rabbit/mouse polyclonal antibodies were produced in our own laboratory. All the other antibodies used in this study were commercial antibodies: anti-β-actin, anti-α-tubulin (Sigma, USA); anti-GFP (Abcam, UK); anti-Myc (Merck Millipore, Germany).

For immunoblotting, the protein samples were heated for 10 min as indicated. The primary antibodies anti-Myc (TMD0^TAP1, TAP2) (clone 4A6, Merck Millipore), anti-C8 (TAP1), anti-tapasin (clone 7F6)18 (link), or β-actin (clone AC-74, Sigma-Aldrich) were used. Blots were incubated with Clarity Western ECL reagent (BioRad) and chemiluminescence was recorded with a Lumi-Imager (Roche) and analyzed with LumiAnalyst software. The signals of the immunoblots were quantified by using ImageJ (NIH).

Co-immunoprecipitation analysis was performed by co-infiltrating N. benthamiana leaves (Kapila et al., 1997 (link); D’Aoust et al., 2009 (link)) with the expression vectors for ARPF2-GFP and ARRS1-MYC. Three days after the infection, the infected leaves (∼100 mg) were frozen in liquid nitrogen and homogenized with zirconia balls in 400 μl of extraction buffer that contained 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% NP40, 10% glycerol, and protease inhibitor cocktail from Sigma-Aldrich Co. LLC (P9599 and 11697498001). The cell lysates were incubated with anti-GFP antibody (MBL Co., Ltd.) and protein A agarose beads (Sigma–Aldrich Co. LLC), and then the proteins interacting with the antibody were eluted with the SDS sample buffer (BioRad, Inc.) and resolved by SDS-PAGE. Following transfer to polyvinylidene difluoride membranes, the proteins were detected with anti-GFP (MBL Co., Ltd., 1:1,000) and anti-MYC (Merck Millipore, 1:1,000) antibodies. In a control experiment, ARRS1-MYC protein was transiently expressed alone or with ARPF2-GFP or GFP-RPL4A in N. benthamiana leaves. Immunoprecipitates obtained with anti-GFP antibody in the experiment were analyzed with anti-MYC and anti-GFP antibodies.

For immunoblot analysis, protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes and detected by Western blot analysis using standard procedures. The antibodies used were peroxidase-conjugated anti-β-actin (Sigma, A3854; diluted to 1:10 000), anti-α-tubulin (Santa Cruz, sc-53030; diluted to 1:1000), anti-FLAG (Sigma, F3165; diluted to 1:1000), anti-myc (EMD Millipore 05-724; diluted to 1:1000), anti-HA (Biolegend, 901501, diluted to 1:1000) and anti-OspB (diluted to 1:10 000). The rabbit anti-OspB antibody was generated (Covance Inc.) against a 14-mer peptide of OspB located 18 residues from the C-terminus.

PK and Pefabloc (a PK inhibitor) were purchased from Roche and Lipofectamine LTX Plus reagent from Invitrogen. Unless stated otherwise, all other reagents and chemicals were obtained from Sigma-Aldrich. Primary antibodies were purchased as follows: anti-HA (Abcam), anti-myc (EMD Millipore), anti-BiP (Santa Cruz Biotechnology), anti-CHOP (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich). The anti-PrP mAb 4H11 has been described previously (63 (link)). Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (goat anti-mouse HRP and goat anti-rabbit HRP).

Cells were grown in YE medium and chromatin isolation and immunoprecipitation were carried out as previously described (Sanso et al., 2011 (link)), with minor modifications. Briefly, cells from 50-mL cultures were cross-linked with 1% formaldehyde for 10 (Clr3-Myc), 15 (Atf1-HA and H3K4me3) or 20 min (H3K9me2 and H3K9ac). Crosslinking was stopped with 125 mM glycine and after lysis of pellets with a bead beater, the lysates were sonicated in order to obtain chromatin fragments of ∼400 bp average size. Once the chromatin was isolated, it was immuno-precipitated with specific antibodies [5 μL of anti-HA antiserum (12CA5; house-made), 1 μL of anti-Myc (Merck Life Science, C3956), 1 μL of anti-H3K9me2 (Abcam, Ab1220) or 1 μL of anti-H3K9ac (Millipore) overnight at 4 °C rotating. Beads were washed, DNA was eluted and formaldehyde cross-linking was reversed. After protein digestion and chromatin extraction, DNA was amplified by quantitative PCR using Light Cycler 480 SYBR Green I Master (Roche). The error bars (SD) were calculated from at least three biological replicates, unless indicated otherwise. Primers from a mitochondrial DNA region or from act1 ORF gene was used as a negative control, as indicated, and they are listed in Table S2.