Enhanced chemiluminescence detection reagent

Cells were lysed with 0.5% NP-40 in PBS supplemented with protease inhibitors for 1 h at 4 °C. After pre-clearing lysates with protein G-Sepharose (Sigma-Aldrich), primary antibodies and then protein G-Sepharose were added to the supernatants and incubated at 4 °C. The protein G-Sepharose beads were washed three times with 0.1% NP-40/PBS. Proteins were eluted from the beads by boiling in 2 × Denature buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 5% β-mercaptoethanol). Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto a polyvinyl difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and probed with the appropriate antibodies overnight at 4 °C. Membranes were washed three times with PBS containing 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were visualized with enhanced chemiluminescence detection reagents (Advansta, Menlo Park, CA, USA). Uncropped images of western blots are shown in Supplementary Fig. 10.

Mycoplasma infected A549 cells were lysed in ice-cold immunoprecipitation buffer (150 mM NaCl, 1% NP 40, 0.5% deoxycholate, 0.1% SDS, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 2 μg/ml aprotinin, 100 μg/ml PMSF, 5 μg/ml leupeptin, 1mM NaF and 1mM NaVO₃) at 4°C for 30 min. Cell lysates and GST-p37 proteins were boiled for 10 min with SDS-PAGE sample buffer. Purified GST-p37 fusion proteins were also treated with 4M urea for 10 min at RT before the treatment of SDS sample buffer. The cell lysates and GST-p37 proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was performed, as described previously [14 , 15 (link)]. The membrane was blocked by 5% skim milk in Tris-buffered saline with 0.1% Tween 20 at RT for 1 hour and incubated with CA27 and α-GST antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour. The membranes were incubated with anti-mouse immunoglobulin G (IgG)-horseradish peroxidase at RT for 1 hour. The proteins were visualized by enhanced chemiluminescence detection reagent (Advansta, Menlo Park, CA, USA).

Proteins were separated with 8.5% SDS-PAGE and transferred to polyvinylidene
difluoride (PVDF) membrane. After blocking with 5% nonfat dry milk containing
TBS-0.1% Tween 20 buffer, membrane was probed with antibodies in the blocking
buffer for overnight at 4°C. For detection of FLAG-GR, CCRP-V5, and EYFP-26/76,
horseradish peroxidase (HRP) conjugating anti-FLAG antibody (1:5000, A8592,
Sigma), anti-V5 antibody (1:5000, 46-0708, Invitrogen), and anti-green
fluorescent protein (GFP) antibody (1:10000, ab6663, Abcam, Cambridge, UK) were
used, respectively. Protein bands on membrane were visualized using enhanced
chemiluminescence detection reagent (Advansta, Menlo Park, California).

Cells were lysed with 0.5% nonyl phenoxypolyethoxylethanol (NP-40, Sigma) in phosphate-buffered saline (PBS) or 1% digitonin with protease inhibitor cocktail (Roche) for 1 h at 4 °C. Cell lysates were incubated with primary antibodies overnight at 4 °C, and then protein G-Sepharose beads (Sigma) were added to the samples for 1–1.5 h at 4 °C. The beads were washed twice with 0.1% NP-40 in PBS. Proteins were eluted by boiling in 2× denaturing buffer (50 mM Tris-HCl, pH 6.8, 2% sodium dodecylsulfate [SDS], 5% β-mercaptoethanol). To completely elute and denature Tom20–Tom70 proteins, 2× denaturing buffer supplemented 15% β-mercaptoethanol was used. The samples were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), blocked with 5% skim milk in PBS containing 0.1% Tween-20 (BioShop Canada Inc., Canada) (PBS-T) for 1 h, washed three times with PBS-T, and probed with appropriate primary antibodies overnight at 4 °C. Membranes were washed three times with PBS-T, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing three times with PBS-T, the immunoblots were visualized using enhanced chemiluminescence detection reagent (Advansta, Menlo Park, CA, USA). Uncropped images of western blots are shown in Supplementary Fig. 6.