Gelcode blue stain

Gastrocnemius muscles were fixed in neutral formalin. For tumour‐bearing mice, non‐tumour side muscle was used. The HTRC core facility of the University of Chicago sectioned the fixed tissues and stained them with haematoxylin–eosin stains. Imaging and quantitation were carried out using Perkin Elmer's Pannoramic Scanner with Pannoramic viewer software (3dhistec Ltd., USA). Tissue lysates for gastrocnemius muscle from non‐tumour side were prepared using RIPA buffer, and expression levels of proteins were checked by immunoblotting 25–30 μg of lysates with indicated antibodies as previously described.⁵ Total protein loading was checked by Coomassie staining of blots using GelCode blue stain (ThermoFisher). Sources of antibodies used in this study are provided in Table2. Immunoblots were quantified using Image J (NIH) and Quantity one (BioRad) software.

Traditional co-IP was carried out as previously described [61 (link)]. For denaturing co-IP of crosslinked proteins, irradiated parasites were lysed in a 1% SDS/50 mM Tris, pH 8.0/150 mM NaCl buffer and boiled at 100°C for 10 minutes to completely denature protein complexes. The lysate was centrifuged, and the supernatant was diluted 10-fold to RIPA conditions prior to IP. Precipitated proteins are either eluted in sample buffer or by high pH with a 100 mM triethylamine solution and dried using a vacuum concentrator. Colloidal Coomassie staining was accomplished using GelCode Blue Stain (ThermoScientific). Gel slices were excised and processed for mass spectrometry. IPs were performed using rat anti-HA (Roche) or mouse anti-V5 (Sigma) agarose beads.

RKO and/or MCF-7 cells were treated with DMSO (vehicle control) or 20 μM inhibitor IV and the cells were harvested with IP lysis buffer. Protein extract was obtained and total protein concentration was estimated using BCA method (Thermo Scientific). Immunoprecipitation was performed for MeCP2 (Sigma, M9317) or species-matched rabbit IgG control, overnight at 4°C. Protein A Dynabeads (Invitrogen) were added the following day to the immune-complex and incubated again for 2.5 hours at 4°C. Immunoprecipitates were washed four times, denatured and SDS-PAGE was performed. The gel was fixed in fixing solution (methanol-acetic acid solution prepared in milliQ water (40% methanol, 10% acetic acid)) and stained overnight using Gelcode Blue stain (Thermo Scientific). Sample bands corresponding to appropriate molecular weight mark for MeCP2 were excised and shipped to Applied Biomics Inc. (Hayward, CA) for acetylation site identification by mass spectrometry.

AlbAB interactions with AlbC and tRNA were first tested by adding mixed samples to NativePAGE loading dye (Invitrogen, Cat# BN20032) followed by loading onto a 1.5% agarose gel prepared with fresh 1x TAE buffer. The agarose gel was run for 50 min at 90 V and 4 °C. tRNA was visualized by staining the gel for 30 min at room temperature with a 1x mixture of GelCode Red (EMD Millipore, Cat# SCT123). Protein was subsequently visualized by staining with GelCode Blue stain (Thermo Scientific, Cat# 1860957) for 18 h and then de-staining with water for an additional 18 h.
To further test whether AlbAB interacts with AlbC or tRNA, mixed samples were prepared containing 10 μM of all components. Mixed samples were then subjected to size-exclusion chromatography using a Superdex S-200 column. AlbAB was found in the void fractions and as such, the void fractions were collected from each run and concentrated to 50 μL. 7.5 μL of each concentrated sample were then analyzed by SDS-PAGE using NuPAGE 4–12% Bis-Tris gels. AlbAB-containing samples were then diluted to 0.05 mg/mL and visualized by negative stain TEM.

Aspartic proteases were affinity purified from the M44 culture supernatant using pepstatin A attached to agarose (Sigma #P2032). The supernatant from 9 days of cultivation contained 23 g/L total protein. The supernatant (15 ml) was batch bound to the resin in 35 ml buffer containing 50 mM sodium acetate and 0.2 M NaCl at pH 3.0. The column was washed with the same binding buffer and bound protein was removed with elution buffer (50 mM Tris-HCL, 1 M NaCl, pH 8.5). Fractions of 0.5 ml were collected. 30 μl of each fraction was mixed with 6 μl of Laemmli sample buffer containing β-mercaptoethanol. The samples were heated at 95°C for 5 minutes before being loaded into a 4–15% PAGE gel (BioRad mini-protean TGX precast gel) along with a low range prestained molecular weight marker (BioRad). The gel was run in SDS PAGE running buffer for 30 minutes at 100 V and then stained with GelCode blue stain (Thermo Scientific).
The 42 kDa doublet band was excised from the SDS PAGE gel and subjected to in-gel trypsin digestion with sequencing grade modified trypsin (Promega #V5111). The resulting peptides were then extracted from the gel and purified by C18 ZipTip (Millipore #ZTC18M096). The purified peptides were analyzed by LC-MS/MS on a QSTAR Pulsar, ESI-hybrid quadrupole-TOF (AB Sciex).