Bp337

All solutions were made in PBS (Life Technologies Gibco® 70013). All wash steps were performed three times using 0.1% Tween 20 (Fisher Scientific BP337) in PBS. Antibodies were diluted into 2.5% BSA (Jackson ImmunoResearch Laboratories 001-000, West Grove, PA) After treatment, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences 15710, Hatfield, PA) for 10 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich 93443) for 10 min, and then washed. Samples were incubated overnight at 4 °C with primary antibodies: β-catenin (1:100 dilution, BD Biosciences 610154), Smad2/3 (1:1000, Cell Signaling Technology 8685), and pSmad1/5/8 (1:100, Cell Signaling Technology 9511). Samples were then washed, stained with 1:1000-diluted secondary antibodies (Alexa Fluor 488/546 αRabbit/αMouse, Life Technologies A11008/A11003) and 2.5 μg/ml Hoechst for 2 h, and washed again. Secondary antibody alone was added to empty wells to serve as references for estimation of uneven illumination.

Sections were pre-treated with Tween-20 (BP337; Fisher Scientific, UK) (0.05%) (wt/vol.) in citrate buffer (10 mmol/l) for 2 h at 65°C and blocked in 3% (wt/vol.) BSA (A7906; Sigma-Aldrich, Madrid, Spain) and 0.5% (wt/vol.) Triton X100 (BP151; Fisher Scientific) for 1 h. Sections were incubated with anti-Iba1 (ionised calcium binding adaptor molecule 1) (ESM Table 1) and anti-GFAP (glial fibrillary acidic protein) (ESM Table 1) antibodies overnight at 4°C in 3% (wt/vol.) BSA and 0.5% (wt/vol.) Triton X100 followed by incubation with secondary antibodies (diluted in PBS) (ESM Table 1) for 1 h at room temperature.

Western blotting was performed as previously described (29 (link)). In brief, after indicated treatments, cells were trypsinized, harvested, and washed with 1X PBS. Pellets were lysed and protein concentrations were determined by the Bradford Assay (Bio-Rad Laboratories, 5000205). Protein samples were loaded and subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and blocked with 5% milk in 1X PBS with 0.1% Tween 20 (Fisher, BP337). The following primary antibodies were used: SQSTM1/p62 (Cell Signaling Technology, 5114T); c-Myc (Cell Signaling Technology, 5605); ATG5 (Cell Signaling Technology, 2630); LC3B (Cell Signaling Technology, 3868); BRD4 (Cell Signaling Technology, 13440S); B-actin (Cell Signaling Technology, 4970); and GAPDH (Cell Signaling Technology, 2118). The membrane was incubated overnight at 4°C with the indicated primary antibodies at a dilution of 1:1000 in 5% BSA. Secondary antibodies: Horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, anti-mouse, 7076S; anti-rabbit, 7074S). The membrane was then washed, secondary antibody was added at a dilution of 1:2000 in 5% BSA for 2 h at room, and the membrane was washed again with 1X PBS with 0.1% Tween 20 three times. Blots were developed using Pierce enhanced chemiluminescence reagents (Thermo Scientific, 32132) on BioRad ChemiDoc System.

Embryos were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710, Hartfield, PA, USA) overnight. Samples from cryosection were rinsed in Tris-Buffered Saline (TBS) (Fisher Chemical, BP2471-1, Hampton, NH, USA) with 0.1% Triton X-100 (Sigma, T9284, St. Louis, MO, USA). Samples were then blocked for 1 h at room temperature in 5% non-fat milk, 0.1% Triton X-100, TBS. Embryos were then incubated at 4 °C overnight for LRP1 (Abcam, ab92544, 1:200, Cambridge, UK) and AP2α (DHSB, 3B5, 1:25). Post antibody incubation was washed with TBS with 0.1% Tween-20 (Fisher Scientific, BP337, Hampton, NH, USA). Secondary incubation of DAPI (Thermo Scientific, 62248, Waltham, MA, USA), donkey anti-rabbit Alexa Flour 555, and donkey anti-mouse 488 took place at room temperature for an hour. Samples were rinsed three more times with TBS with 0.1% Tween-20, then mounted on SuperFrost Plus Microscope Slides (Fisher Scientific, 12-550-15, Hampton, NH, USA) with ProLong Glass Antifade Mountant (Invitrogen, P36982, Waltham, MA, USA). Embryos were then imaged using Leica TCS SP8 (Wetzlar, Germany).

The proximity ligation assay (PLA) was performed using the Duolink Red Starter Kit for Rabbit/Mouse (Sigma #DUO92101) according to the manufacturer’s specifications. Briefly, cells were fixed with 3.7% formalin (Sigma-Aldrich #F8775) for 25 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich #X-100) for 5 min, and blocked using 0.05% Tween20 (Fisher Scientific #BP337) in 2mg/mL in PBS for 1 hour (all steps performed at room temperature). Samples were then incubated with primary antibodies against acetyl-p53 K373 (EMD Millipore #04–1137) and Bax (Santa Cruz #sc-7480), followed by incubation with the PLA probes. The Duolink PLUS and MINUS probe was used against the anti-acetyl-p53 K373 and anti-Bax antibodies, respectively. If the two proteins are interacting, the oligonucleotide PLA probes would be in proximity of each other. Ligation of the co-localized probes was performed using a ligase and amplification of the ligated oligonucleotides was achieved through rolling circle replication with a polymerase. Samples were then incubated with fluorescent probes specific for the oligonucleotides. Slides were imaged on the Zeiss Elyra PS1 Super-Resolution Microscope using structured illumination (SIM) at the Delaware Biotechnology Institute Bioimaging Center.

DNA in vitro transcription template array (prepared according to methods and approaches Section 2.5).
Streptavidin-coated RNA capture surface (prepared according to methods and approaches Section 2.4).
Parafilm (Bemis Flexible Packaging).
MEGAscript T7 Transcription Kit (Invitrogen; AMB1334-5).
PBS (Fisher Scientific; BP399).
Tween 20 (Fisher Scientific; BP337).