Ai600

In vitro transcription assays were performed as previously described (Su et al., 2016 (link)). Promoter sequence fragments (311 bp) of hrpX were acquired using PCR with the primer set hrpXivt‐F/R (Table S3). The obtained hrpX promoter sequence fragments and HrpG or/and VemR protein were incubated for 30 min at room temperature in transcription buffer. Then, a NTP mixture (250 μM each of ATP, CTP, and GTP, 250 μM biotin‐16‐UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs) were added to initiate transcription. After incubation at 28°C for 30 min, the reactions were terminated and the transcription products were analysed by electrophoresis. The transcripts obtained were visualized using a phosphor imager screen (GE AI600).

To detect the K48 polyubiquitination of HIF1α in cells, we co-transfected Flag-RBCK1/Flag-tag and Myc-HIF1α and K48-Ub plasmids in HEK293T. 48 h after transfection by a western blot and IP technology, we obtained the corresponding protein supernatant. Finally, we visualized the fluorescent signal of the resulting protein using AI600 (GE), during which the membrane was pre-processed with an Immobilon Western Chemilum HRP Substrate Kit (Millipore Co, Billerica).

Cells were plated in 6-well dishes, drug-treated (see Figure legends for treatment times), washed, incubated for an additional 4–6 days, fixed (50% methanol), and stained (0.05–0.1% crystal violet in 50% methanol). The plates were imaged on a GE Healthcare AI600, and colonies/staining were quantitated with the ImageJ/ColonyArea Plug-In [20 (link)].

To obtain proteins of HL-1 cells or cardiac tissues, a RIPA lysis buffer (89901; Thermo Fisher, IL, USA) containing 1% protease and phosphatase inhibitor (78442; Thermo Fisher, IL, USA) was used to lyse cells and cardiac tissue. Protein concentration was quantified using a BCA assay kit (23225; Thermo Fisher, IL, USA). After quantification, 5× sodium dodecyl sulfate (SDS) protein loading buffer (P0015L; Beyotime, Shanghai, China) was mixed with the protein lysates, and then the protein mix was denatured at 95°C for 10 min. The same amount of protein was loaded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, CA, USA). The PVDF membrane was blocked with a blocking buffer (P0252; Beyotime, Shanghai, China) at room temperature for 30 min, followed by incubating with a primary antibody (β-tubulin and MyD88 (2146 and 4283, Cell Signaling Technology, MA, USA) antibody) at 4°C overnight. The next day, the membrane was detected with a chemiluminescence system (GE AI600, MA, USA) after incubating with the HRP-conjugated secondary antibody.

To extract proteins from flies of different developmental stages, larvae, pupae, and adult flies were collected and frozen with liquid nitrogen for 1 min. For protein extraction from different tissues, the tissues were dissected in Grace’s Insect Medium (Life) and then were frozen with liquid nitrogen for 1 min. Samples were prepared with RIPA lysis buffer and 1X protease inhibitor cocktail (Bimake) and then ground for 10 min. Following 20 min incubation on ice, samples were centrifuged at 13,000 rpm at 4 °C for 30 min. The supernatants were collected and boiled with 1X protein loading buffer at 95 °C for 10 min. Lysates were separated by 4–20% SDS-PAGE gels, followed by transferring to PVDF membranes (Roche). After blocking with 5% BSA in TBST for 1 hour, membranes were incubated with HRP-conjugated antibody (1:3000, Cell Signaling, Cat. # 3999 s) for 1 hour. After three times washing in TBST, biotinylated proteins were visualized using the enhanced chemiluminescence system AI600 (GE). To detect the recombination expression of ligase, blocked membranes were incubated with anti-V5 antibody (1:3000, Invitrogen) at 4 °C overnight. Following three washes in TBST, membranes were incubated with HRP-linked anti-mouse antibody (1:3000, Cell Signaling, Cat. # 7076 s) for 1 hour. Then, membranes were washed with TBST three times and visualized by the enhanced chemiluminescence system.

NPDF lysates were collected in lysis buffer (G-Biosciences, St. Louis, MO, USA) with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The same amount of proteins was separated using 10% sodium dodecyl sulfate-polyacrylamide mini-gel electrophoresis and transferred to the nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK). After the night-time incubation of a specific primary antibody (α-SMA, Col-1, fibronectin, and p-Smad 2), the membrane was incubated with a secondary antibody (IgG for goat anti-mouse) conjugated to horseradish peroxidase. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL, USA). Band images were captured and analyzed using image systems (AI 600, GE Healthcare Life Sciences, Canton, MA, USA) and ImageJ software (ver. 1.52a; National Institutes of Health, Bethesda, MD, USA).

A subset of animals (n = 5/group) had cytosolic and nuclear levels of Cited2 protein determined. SDS-PAGE and immunoblotting protocols were similar to previous studies from our lab [12 (link), 28 ]. Briefly, ~25 mg of gastrocnemius muscle was homogenized and separated into cytosolic and nuclear fractions by a commercially available kit according to the manufacturer’s instructions (NE-PER Nuclear and Cytoplasmic Extraction Kit; Pierce). Protein was quantified by a microBCA kit (Pierce). 60 μg of protein was separated by SDS poly-acrylamide gel and transferred onto a PVDF membrane. After transfer, membranes were blocked with 5% non-fat dry milk in Tris buffered saline, 0.01%Tween (TBST) for 1 h and incubated overnight at 4°C with antibodies against Cited2 (Abcam), ß-tubulin or Histone H3 (Cell Signaling) diluted 1:1000 in TBST /1% non fat dry milk. Membranes were rinsed 3 times for 10 min with TBST and incubated with an anti-rabbit horseradish peroxidase conjugated secondary antibody (Cell Signaling) diluted 1:2000 in TBST 1% milk for 1 h then rinsed 3 times for 10 min. Membranes were then incubated with a chemiluminescent solution (ECL; GE Healthcare) for 5 min and imaged using a CCD digital camera system (AI600, GE Healthcare). Images were quantified using ImageQuant 8.0 (GE Healthcare)