Anti rabbit

The cryocut brain sections from various stages of development in utero and postnatal life (E11, E14, E16, E18, P0, P2, P6, P15, P21 and P30) were air dried and then washed in PBS. The membrane permeabilisation was achieved by treating sections with 1% Triton X-100 in PBS for 20 min. The sections were subsequently washed thrice with PBST (0.5% Tween-20 added to PBS) and then incubated for 2 h with 10% normal goat serum (NGS) in PBS at room temperature for non-specific protein blocking. After blocking the sections were incubated overnight at 4°C with primary antibodies, i.e. anti- A₂B₅ (1:200, Mouse monoclonal, Abcam ab53521) or anti-BLBP (1:300, Rabbit polyclonal, Abcam ab32423). The binding of the primary antibodies, i.e. anti-A₂B₅ and anti-BLBP was visualized using goat raised TRITC labelled anti mouse (1:200, Sigma) and anti-Rabbit (1:200, Sigma) antibodies respectively. Both the primary and secondary antibodies were diluted in 5% BSA in PBS with 0.5% Tween-20. Control for immune labelling was performed with the same procedure without the primary antibodies. The sections after thorough washing with PBS were finally cover-slipped with Vectashield Hard+Set mounting medium with DAPI and visualized under the fluorescence microscope.

HEK293FT cells (ATCC, Manassas, VA, USA; #CRL-3216) or HEK293T cells (ATCC, Manassas, VA, USA; #CRL-1573) were cultured in 6-well plate and transfected with 1.5 μg Flag-p62 and 1.5 μg HA-ATF2 plasmid per well by polyethylenimine (linear, MW-25000, Polysciences, Hirschberg, Germany) for 24 h. Isoproterenol (10 μM) was added half an hour before the cells were collected. The protein lysate of 1500 μg was incubated with anti-Flag-agarose (Sigma-Aldrich, Munich, Germany) overnight for immunoprecipitation, together with 50 μg lysate as input to be loaded for western blotting detection of HA (anti-HA, Cell Signaling Technology) or Flag tag (anti-Flag). Antibodies were obtained from the following sources: anti-HA (C29F4) (Cell Signaling, Danvers, MA, USA; #3724S; dilution: 1:1000), anti-FLAG-M2 (Sigma-Aldrich, St. Louis, MO, USA; #F1804; dilution: 1:2000), anti-beta-actin (Cell Signaling, Danvers, MA, USA #4967S; dilution: 1:2000), anti-rabbit (Sigma-Aldrich, St. Louis, MO, USA; #A3687; dilution: 1:20,000), anti-mouse (Sigma-Aldrich, St. Louis, MO, USA; #A3562; dilution: 1:20,000).

Dulbecco’s Modified Eagle’s Medium (DMEM) and other cell culture supplies, such as trypsinogen, were purchased from Invitrogen. Fetal calf serum was purchased from Gibco BRL (Grand Island, NY, USA). A Cell Counting Kit-8 was purchased from Dojindo (Dojindo Laboratories, China). HO-1 and pAbR rabbit antibody were purchased from Enzo Life Sciences Inc. (Ann Arbor, Mich., USA). p62 and LC-3 antibody were purchased from Abcam (USA), and GAPDH antibody was obtained from CST (USA). AntiRabbit was obtained from Sigma (USA). BSA was obtained from Invitrogen (USA). TritonX-100 was obtained from Sigma (USA). A JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide) Mitochondrial Membrane Potential Detection Kit was purchased from Beyotime (China). Confocal microscopy and imaging systems (ZEISS-Axio) were used at the Wuhan University School of Basic Medical Sciences.
Zinc protoporphyrin (ZnPP), a HO-1 inhibitor, was supplied by Sigma. The purity of ZnPP (≥95%) was determined by HPLC; the inhibitor was soluble in water and has the molecular formula C₃₄H₃₂N₄O₄Zn and a molecular weight of 626.03 g/mol.