Anti flag m2 f3165

HEK293 (ATCC CRL-1573) cells and their derivative cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. PGAP1-KO^{69 (link)} and PGAP2-KO^{70 (link)} cells have been constructed previously. KO cell lines used in this study are listed in Supplementary Table 1. Mouse monoclonal anti-CD55 (clone IA10)^{50 (link)}, anti-CD59 (clone 5H8)^{50 (link)}, anti-CD230 (14-9230-82; Thermo Fisher Scientific), anti-CD109 (556039; BD Biosciences), anti-GAPDH (60004-1-Ig, clone 1E6D9; Proteintech), anti-FLAG (F3165; M2; Sigma-Aldrich) and rabbit monoclonal anti-HA (3724S; Cell Signaling Technology) were used as primary antibodies. F(ab’)2-goat anti-mouse IgG, PE (12-4010-82; Thermo Fisher Scientific) and F(ab’)2-donkey anti-rabbit IgG, PE (12-4739-81; Thermo Fisher Scientific), and Goat Anti-Mouse IgG, HRP (HS201-1; TransGen Biotech) were used as the secondary antibodies. For flow cytometric analysis, antibodies were used at 10 µg/ml. For western blotting, the primary antibodies and the secondary antibodies were used at 1 and 0.2 µg/ml, respectively. PI-PLC (Thermo Fisher Scientific) and proaerolysin (produced and purified in the laboratory) were used for treatments. Cell Counting Kit-8 (CCK-8, MedChemExpress) was used to detect cell viability.

The rabbit polyclonal antibody against RTTN was raised using recombinant RTTN-His (residues 1347–1591) and subjected to affinity purification. The following antibodies were used in this studies: antibodies against SAS-6 (residues 1–489, 1:500 dilution)^{13 (link)}, CPAP (residues 1070–1338, 1:1000 dilution)^{50 (link)}, CEP120 (residues 639–986, 1:1000 dilution)^{15 (link)}, CEP135 (residues 650–1140, 1:1000 dilution)^{9 (link)}, CEP295 (residues 2092–2430, 1:500 dilution)^{19 (link)}, centrobin (residues 443–626, 1:1000 dilution)^{13 (link)}, and centrin 2 (residues 1–173, 1:1000 dilution)^{13 (link)}. The other commercially available antibodies used in this study included anti-STIL (A302–442; 1:200 dilution), anti-hPOC5 (A303–341; 1:500 dilution) and anti-SPICE (A303–272; 1:500 dilution) (all from Bethyl); anti-hSAS-6 (H00163786; 1:100 dilution, monoclonal Ab), anti-centrin3 (H00001070-M01; 1:1000 dilution) and anti-CEP162 (PAB22408; 1:1000 dilution) (all from Abnova); anti-CP110 (12780-1-1p; 1:500 dilution; Proteintech); anti-CEP164 (NBP1-81445; 1:500 dilution; Novus Biologicals); anti-POC1B (PA5-24495; 1:500 dilution; Thermo Fisher); anti-acetylated tubulin (T6793; 1:1000 dilution) and anti-Flag (M2; F3165; 1:3000 dilution) (both from Sigma-Aldrich); and anti-GFP (632381; 1:3000 dilution; BD Bioscience).

Transfected cells were harvested with RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, and 1X protease inhibitor cocktail set III (Calbiochem)) 2 days after transfection. Immunoprecipitation was performed with mouse anti-Myc (9E10, Santa Cruz) antibody or anti-Flag-M2 (F3165, SIGMA) antibody. The antibodies were removed by incubation with protein G Sepharose (Zymed/Invitrogen), and the wash step was repeated 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). To examine the interaction of Flag-C16orf74 with Myc-PPP3CA, we analyzed the immune complexes by western blotting with rabbit anti-Flag and anti-Myc antibodies. To examine the interaction of endogenous C16orf74 with endogenous PPP3CA, Capan-1 cells were harvested in RIPA buffer, and immunoprecipitation was performed with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody or rabbit anti-C16orf74 (immunized with full-length recombinant C16orf74 protein) antibodies. Then, we collected the antibodies with protein G Sepharose (Zymed/Invitrogen) and repeated the wash steps 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). We analyzed the immune complexes by Western blotting with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody, or rabbit anti-C16orf74 antibodies.

The following commercial antibodies were used: HRP-conjugated anti-mouse I gG (NA931), and anti-rabbit IgG (NA941) from GE Healthcare (Pittsburgh, PA), HRP-conjugated anti-rat IgG (AP136P), and anti-ubiquitin Lys63 specific (05–1313) from EMD Millipore (Billerica, MA), anti-TAK1 (sc-7162), anti-IKKα/β.(sc-7607), anti-IκBα.(sc-371), anti-JNK (sc-7345), anti-p38 (sc-728), anti-Tab1 (sc-6052), anti-β-actin (sc-69879), and anti-ubiquitin (sc-8017) from Santa Cruz (Dallas, Texas), anti-p-TAK1 (4531), anti-p-IKKα/β.(2078), anti-p-IκBα.(9246),.anti-p-JNK (9251), and anti-p-p38 (9211) from Cell Signaling (Beverly, MA), anti-HA (11867423001) from Roche (Indianapolis, IN), and anti-Flag (M2, F3165) from Sigma (St. Louis, MO). LPS, TNF, IL-1β,.5Z-7-oxozeaenol, dithiothreitol (DTT), iodoacetamide (IAA), and formic acid were obtained from Sigma. Acetonitrile (ACN) and ammonium bicarbonate were obtained from Aldrich (Milwaukee, WI). Sequencing-grade trypsin was purchased from Promega (Madison, WI).

Protein lysates from whole tissues were prepared by homogenising in ten volumes of urea lysis buffer, as described [1 (link)]. Samples were quantified using a BCA assay (Thermo Scientific, VIC, Australia), separated on polyacrylamide gels (Bio-Rad, Gladesville, NSW, Australia) and immunoblotted. Clarity Western ECL substrate was used for visualisation (Bio-Rad, Gladesville, NSW, Australia). Antibodies used were as follows: anti-RLF, Ab115011 and anti-RCOR1, Ab32631 (Abcam, Cambridge, UK); anti-LSD1, 2139 and anti-HP1α, 2616 (Cell Signaling, Danvers, MA, USA); anti-Flag M2, F3165 and anti-γ-tubulin, T5192 (Sigma-Aldrich, St. Louis, MO, USA); anti-MRE11 (12D7) GTX70212, anti-NBS1 (1C3) GTX70222 and anti-RAD50 (13B3) GTX70228 (GeneTex, Irvine, CA).