Ab2851

The binding of HOTAIR to Dnmt3b protein was detected using a RIP kit (Merck Millipore, Billerica, MA, USA). After being washed with pre-cooled and lysed with an equal volume of RIPA lysate (P0013B, Beyotime Institute of Biotechnology, Shanghai, China) in an ice bath for 5 min, the cells were centrifuged at 14,000 rpm for 10 min at 4 °C. Part of the cell extract was taken as input, and remaining cell extract was incubated with antibody for co-precipitation. A total of 50 μL magnetic beads for each co-precipitation reaction system were washed and re-suspended with 100 μL RIP Wash Buffer, and then 5 μg of following antibody was added: rabbit anti Dmnt3b (1:100, ab2851, Abcam Inc., Cambridge, MA, UK) or rabbit anti-human immunoglobulin G (IgG) (1:100, ab109489, Abcam Inc., Cambridge, MA, UK) as an NC. The magnetic bead-antibody complex was washed, re-suspended with 900 μL RIP Wash Buffer, and incubated with 100 μL cell extract overnight at 4 °C. The samples were washed 3 times and placed on magnetic pedestal to collect the magnetic bead-protein complexes. The collected samples and input were separately digested by proteinase K to extract RNA for detecting HOTAIR in subsequent RT-qPCR.

The Soluble protein components of the cells were collected after cell lysis. The protein concentration was determined by the Bradford assay (Bio-Rad). Equal amounts of lysate were applied to a 10% NuPAGE Bis-Tris Gel (Invitrogen) and gel electrophoresis was performed. The lysates were then transferred onto PVDF membranes for 90mins, and then the reactive sites were blocked with the help of a 5% skimmed milk solution. The membranes were washed once with TBST, then incubated for overnight at 4°C with relevant primary antibodies (ACADS, 16623-1-AP, proteintech; DNMT1, 24206-1-AP, proteintech; DNMT3A, 19366-1-AP, proteintach; DNMT3B, ab2851, abcam). The membranes were then washed again for three times with TBST after which the secondary antibody (Goat Anti-Rabbit IgG 1:5000) was added and the samples were incubated for a duration of 2 hours at room temperature. Upon the conclusion of this step, the membranes were subjected to three washes with TBST and then suitable amounts of ECL liquid was added and the membranes were placed in a darkroom for the reaction to proceed. A GAPDH solution (1:5000 dilution, Sigma-Aldrich) was used as the loading control reagent.

ATRA (All-trans-retinoic acid), 5-aza-2-deoxycytidine (DAC), trichostatin A (TSA) and anti-PADI4 (P4874) were purchased from Sigma (St. Louis, MO). Cl-amidine was from Cayman Chemical Company in USA. pGL3-basic vector and Dual Luciferase Reporter Assay System were from Promega Corp. Antibody specific to SOX4 (ab80261), H3R17Me (Ab8284), H3Cit (Ab5103), DNMT1 (ab13537), DNMT3a (ab2850), DNMT3b (ab2851), Tubulin (ab126165), Lambin B (ab16048) and horseradish peroxidase coupled secondary antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-GAPDH (sc-47724) and anti-PU.1 antibody (sc-365208) were from Santa Cruz Biotechnology Anti-human CD11b antibody (11–0113) was from eBiosciences in San Diego of USA.

Bone marrow tissue was EDTA-decalcified, formalin-fixed, and processed into 4 μm sections. Staining with an anti-DNMT3B antibody (Abcam ab2851; 1:250 dilution) was done using an automated stainer (BenchMark Ultra, Ventana-Roche Diagnostics). miR29b was detected in situ using the miRNAscope HD Assay Red (Advanced Cell Diagnostics, Newark (CA), USA) and an miR29b-specific probe (SR-hsa-miR-29b-3p-S1 MIMAT0000100, Advanced Cell Diagnostics). The negative control was a non-specific miRNA probe (Advanced Cell Diagnostics). Slides were digitalized and analyzed on an Aperio GT 450 DX slide scanner (Leica) at 400 × magnification.