Anti ha antibody conjugated agarose

The epitope-tagging strategy to isolate ASPP1 (or ASPP2)-containing protein complexes from human cells was performed essentially as previously described with some modifications [45 (link)]. In brief, to obtain a FLAG-HA-ASPP1 (or ASPP2) expressing cell line, HeLa cells were transfected with pCIN4-FLAG-HA-ASPP1 (or ASPP2) constructs and selected for 2 weeks in 1 mg/ml G418. The tagged ASPP1 or ASPP2 protein levels were detected by WB analyses. The stable cell lines were chosen to expand for protein complex purification. For purification, the MOCK, HeLa/ASPP1, or HeLa/ASPP2 cells were lysed in BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100 and fresh protease inhibitor on ice for 2 hr. The homogenate was centrifuged for 30 min at 12000 rpm at 4°C. Cleared lysates were filtered through 0.45 μM spin filters (Millipore) and immunoprecipitated by anti-FLAG antibody-conjugated M2 agarose (Sigma). The bound polypeptides eluted with the FLAG peptide (Sigma) were further affinity purified by anti-HA antibody-conjugated agarose (Sigma). The final elutes from the HA-beads with HA peptides were resolved by SDS-PAGE on a 4%–20% gradient gel (Bio-Rad) for Coomassie Blue staining. Gel bands were cut out from the gel and subjected to mass-spectrometric sequencing.

For IP of GFP or HA-tagged proteins, anti–GFP antibody–conjugated agarose (ChromoTek) and anti-HA antibody–conjugated agarose (Sigma) were used. For endogenous proteins, 1 μg relevant antibody was pre-incubated with 10 μl protein-G Sepharose packed beads and washed to remove non-bound antibody. 0.5–2.5 mg lysates were precleared with 10–30 μl protein-G Sepharose (GE Healthcare), and then incubated with an antibody–resin conjugate for 2 h at 4°C under gentle agitation. IPs were washed three times with lysis buffer containing 0.1 M NaCl. Typically, 1%–2% input and 30% of IP eluates were loaded for Western blot, except in Figs 1F and 4A–C in which 15% of eluate was loaded. For RNAse treatment, 40 μg RNAse A was incubated with IP or 2 μg HeLa RNA for 60 min at 4°C. Proteins were eluted in SDS-sample buffer. Western blots were performed by standard protocols. CMTR1 antibody was raised in sheep by Orygen Antibodies Limited, UK and affinity purified against the human recombinant protein. Second bleed was used at 1 μg/ml for Western blotting and the first bleed was used at 1 μg/IP. Other antibodies: RNGTT (in-house), HA (Sigma), DHX15 (ab70454; Abcam), GAPDH (Cell Signaling Technologies), RNA pol II (N20; Santa Cruz), PSer5-PolII (3E8; ChromoTek), and pSer2-PolII (3E10; ChromoTek). Secondary antibodies were from Pierce.

Crude mitochondria were isolated and resuspended to a concentration of 5 mg/mL. Mitochondria was solubilized in 0.7% digitonin for 30 min. Followed by centrifugation at 20,000 ×g for 20 min. Cleared mitochondrial lysates were incubated with anti-HA antibody conjugated agarose (Sigma) for 2 hr. at 4°C. The agarose was washed 3–5 times and eluted in Laemmli buffer (65°C, 10 min). Elutions were resolved by SDS-PAGE and assessed by immunoblot.

Mitochondrial isolation and immunoprecipitation were performed as described previously (Van Vranken et al., 2018 (link)). Briefly, cell pellets were washed and lysed as described in the ‘Crude mitochondrial isolation’ section. Spheroplasts were washed once and homogenized in ice-cold SEH buffer (0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.4, 1 mM PMSF, yPIC) with 10 mM N-ethylmaleimide (NEM) (Sigma, E3876) by applying 20 strokes in a dounce homogenizer. Crude mitochondria were isolated by differential centrifugation. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific). 1 mg of crude mitochondria were resuspended in 200 μl of XWA buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, pH 7.4) with 10 mM NEM and 0.7% digitonin added and incubated on ice for 30 min. After centrifugation at 20,000 × g for 20 min, solubilized mitochondria were incubated with pre-equilibrated anti-HA antibody-conjugated agarose (Sigma, A2095) for 2 hr at 4°C. The agarose was washed three times and eluted by incubating in 2× Laemmli buffer at 65°C for 10 min. Elution was isolated by SDS-PAGE and subjected for immunoblot analysis.