Anti ubiquitin antibody

To conduct the in vivo ubiquitination assay, ‘lactate-’ or ‘lactate + ’ culture medium was generated by excluding or adding (respectively) the proteasome inhibitor MG132 (10 μM, Selleck, Berlin, Germany; S2619) from the culture during 8 h of OGD conditions prior to harvesting. Cell lysates were precleared by adding 50 μL of protein A/G immune magnetic beads (Bimake, Houston, TX, USA; B23201) and were immunoprecipitated with anti-NDRG2 antibody. Polyubiquitinated forms of NDRG2 were detected by western blot with an anti-ubiquitin antibody (1:1000, Cell Signaling Technology, A100). Astrocytes were transfected with expression vectors (i.e., different forms of Flag-tagged NDRG2 overexpressing vector [WT, K176A, K176A + R240A]); 72 h later, the medium was changed to a ‘lactate + ’ culture medium by administering MG132 for 8 h under OGD conditions prior to harvesting. Cell lysates were precleared by adding 20 μL of anti-Flag-conjugated magnetic beads (Bimake, B26101) and were subsequently immunoprecipitated with anti-NDRG2 antibody. Proteins immunoprecipitated with anti-Flag magnetic beads were detected by western blot using an anti-ubiquitin antibody.

An anti-IGF2BP2 antibody (1–2 mg per test, Abcam, ab124930) and an anti-FLAG/DYKDDDDK Tag (1–2 mg per test, Cell Signaling Technology, 8146 s) were used in the IP assays, and the proteins were detected by Western blotting with an anti-ubiquitin antibody (1:1000, Cell Signaling Technology, #3933) according to the manufacturer’s instructions.

The WiDr cells were treated with 50 μM SeC for 9 h or 24 h. The cell lysates were prepared in M-PER lysis buffer along with the phosphatase and protease inhibitor cocktail, and then centrifuged at 14,000 rpm for 15 min. Equal amounts of cell lysate were incubated with anti-Nrf2 (GeneTex, GTX103322, 1.3 μg) or anti-Keap1 antibody (ABclonal, A17061, 1 μg) at 4 °C for overnight. The immune complexes were precipitated with protein A-Sepharose beads at 4 °C for 4 h. The beads were washed with 0.2% Tween 20 in PBS and eluted in 150 mM glycine-HCl (pH2.6). The eluted proteins were neutralized by adding 1.7 M NaOH, and then boiled in 4× sample buffer. Proteins were separated by TGX Stain-Free FastCast Acrylamine Kit and immunoblotted with anti-ubiquitin antibody (Cell signaling, #58395, 1000× dilution).

The antibodies and compounds used were commercially obtained: anti-RPE65 antibody (Catalog No. ab13826, Abcam, Cambridge, MA), anti-RNF123 antibody (Catalog No. A09642-1, BOSTER, China), anti-RNF149 (Catalog No. bs-9228R, Bioss, China), anti-ubiquitin antibody (Catalog No. 3933, Cell Signaling Technology, Boston, MA), anti-β-Actin (Catalog No. EM21002, Huabio, China), D-galactose (D-gal, Catalog No. G0750, Sigma-Aldrich, St. Louis, MI), H₂O₂ (Catalog No. 323381, Sigma-Aldrich), DAPI (Catalog No. 9542, Sigma-Aldrich), and lutein (Catalog No. 07168, Sigma-Aldrich).

As previously described method,²⁹ the 293T cells were transfected with plasmids including Myc‐ubiquitin, Flag‐GATA4, HA‐RING1B and/or His‐Bmi‐1, and lysed in the presence of deubiquitination inhibitor N‐ethylmaleimide (E3876, Sigma‐Aldrich, USA), and subjected to immunoprecipitation with anti‐DYKDDDDK Tag (Anti‐FLAG M2 antibody, #14793, Cell Signaling Technology, USA) antibody. Western blot was used to detect the ubiquitination levels with anti‐ubiquitin antibody (#91112, Cell Signaling Technology, USA).

Two micrograms of each protein were loaded and separated by a 10% SDS–PAGE gel. This was electro-transferred to a PVDF membrane. The membrane was incubated with anti-Ubiquitin antibody (Cell Signaling Technology). This was then treated with peroxidase-conjugated anti-rabbit IgG polyclonal antibody (Cell Signaling Technology). The membrane was washed and incubated with chemiluminescent substrate followed by exposure to the photographic film and visualized post development.

Endogenous Sox9 was immunoprecipited using the anti-Sox9 antibody (AB5535, Millipore) in denaturing conditions. The purified Sox9 protein was subjected to Western blot analysis and immunoblotted with anti-ubiquitin antibody (3936, Cell Signaling Technology).