Ubiquityl histone h2a

Antibodies (dilutions are indicated in brackets for western blot (WB), immunofluorescence (IF), or immunoprecipitation (IP)) against FLAG (Sigma, clone M2; Sigma, produced in Rabbit, IP 3 μl/sample, IF 1:100, WB 1:1000), FLAG (Sigma, clone M2; Sigma, M, Wb 1:1000, IF 1:200), ubiquityl-histone H2A (Millipore, clone E6C5), ubiquitin (Norvus Biologicals, FK2, M, WB 1:1000, IF 1:1000; Dako WB), K48-linkage specific polyubiquitin (Enzo lifesciences, WB 1:1000), K63-linkage specific polyubiquitin (Cell Signaling, clone D7A11, 1:1000), myc (MBL, clone PL14, WB 1:3000, IF 1:100), HSC70 (Stressgen, WB 1:5000, IF 1:100), LC3B (Novus Biologicals, NB100-220), GFP (clonetech, 632381), p62 (Enzo Life Sciences, BML-PW9860), Lamin A/C (Santa Cruz, 4A58), HSC70 (Stressmarq biosciences), HSP70 (Stressgen, clone SPA-810, WB 1:1000, IF 1:50), HSPA1A (Enzo life sciences, Rb, WB 1:1000), HSPB1 (Stressmarq biosciences), GAPDH (Fitzgerald, clone 6C5, WB 1:50,000), histone H2A (Abcam, WB 1:5000), MYC (Clonetech, Mountain View, CA, USA), and DNAJB1/Hsp40 (Stressgen, San Diego, CA, USA, Rb, 1:1000) were used.
MG132 (20 µM for 3–6 h), rapamycin, Pepstatin A (10 μg/ml), E64d (10 μg/ml), 3-methyladenine (3-MA, 10 mM) ammonium chloride (NH₄Cl, 20 mM) were from sigma.

CUT&RUN-qPCR is a new method to extract the DNA bound to protein. NSCs stably expressing Mysm1-Flag and induced astrocytes were harvested and treated according to the manufacturer’s instructions by using a Hyperactive pG-MNase CUT&RUN Assay Kit for PCR/qPCR (HD101, Vazyme Biotech). In brief, cells were collected to combined with ConA Beads Pro. Subsequently, cells were resuspended in antibody buffer and incubated with primary antibodies against Flag (F1804, Sigma‒Aldrich), ubiquityl-Histone H2A (#05-678, Millipore), Tri-Methyl-Histone H3 (Lys4) (#9751, Cell Signaling Technology), or Acetyl-Histone H3 (Lys9) (#9649, Cell Signaling Technology). Antibody-protein complexes were incubated with pG-MNase Enzyme. Then fragmentation was terminated and released by Stop Buffer containing Spike in DNA. The DNAs were extracted by FastPure gDNA Mini Columns and subjected to qPCR. The Spike in DNA was used as a reference and the primer sequences for CUT&RUN-qPCR are listed in Supplementary Table 3. All cells were tested for mycoplasma contamination before experiment.