Ab32106

Chromatin immunoprecipitation (ChIP) was performed using Millipore Chip Kit (catalog #17-10085) and procedures were according to the manufacturer’s protocol and a previously study^{61 (link)}. Shortly speaking, cells cultured under the previously indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 mL of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from the soluble chromatin preparations were 400–800 bp in length. Immunoprecipitation was carried out overnight with purified anti-ELK1(abcam, ab32106), anti-NFκB p65 (abcam, ab19870) or normal rabbit IgG as a negative control. Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed using 2 μL DNA samples with the following primers: LRG-1 primer: forward, 5′-TGTCACTACATTTCACAAGCCT-3′; reverse, 5′-CCAGCCGTTAGTTGGTCTTA-3′

ChIP-IT PBMC kit (cat no. 53042 from Active Motif) was used according to the manufacturer’s instructions. D3-activated naive B cells primed with IL-2 were fixed according to the protocol and lysed by sonication using the Epishear probe sonicator and the cooled sonication platform (Active Motif). Sonicated chromatin was controlled on gel prior to immunoprecipitation. Rabbit antibodies to ELK1 (ab32106), H3K9ac (ab4441), H3K27ac (ab4729), H3K4me1 (ab8895), p300 (ab10485) and IgG (ab37415) were from Abcam. Results were analysed with the ChIP-IT qPCR Analysis kit (cat no. 53029 from Active Motif) to calculate binding events detected per 1000 cells. Human Negative Control Primer Set1 and Human Positive Control Primer Set GAPDH⁻2 from the ChIP-IT qPCR Analysis kit were used, other primers are listed in Supplementary Table 4.

Tissue sections (5 μm) derived from xenografted tumors in nude mice were immunostained with CD34 (ab81289, Abcam Inc.), ki67 (ab16667, Abcam Inc.), and ELK1 (ab32106, Abcam Inc.). The mean intensities for positive expression of each protein product were determined by using the software Image-ProPlus 6.3 (Media Cybernetics, USA). Brown stains were defined as positive staining. The mean positive expression rate was obtained from five randomly selected fields, with a total of 200 cells in each field.

The osteosarcoma cells were immobilized with formaldehyde for 10 min to produce DNA-protein crosslinks. Ultrasound (UP-250, Ningbo Scientz Biotechnology Co., LTD., Ningbo, Zhejiang, China) was used to break the cell chromatin into fragments for 15 cycles, 10 s one time at intervals of 10 s. Next, the samples were centrifuged at 12000 rpm for 10 min to collect supernatant, which was later divided into two tubes, and incubated with negative control (NC) rabbit IgG (ab109489, 1: 300, Abcam) and ELK1 antibodies (1: 100, ab32106, Abcam) respectively at 4° C overnight. Protein A Agarose/SaLmon Sperm DNA was added to the cells and centrifuged at 12000 g for 5 min to remove the supernatant. After the non-specific complex had been washed off, the complex was de-crosslinked at 65° C. Phenol/Chloroform was used to extract and purify the recovered DNA fragments. The binding of ELK1 to miR-134 was detected by RT-qPCR with specific primers of miR-134.

Halo-TDP-43 i3Neurons were homogenised in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% Glycerol, 2 mM EDTA, 0,1% SDS, protease inhibitor (cOmplete^™ EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitor (PhoSTOP^™, Roche)). Samples were loaded on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen), which was run in NuPAGE MOPS buffer. Proteins were transferred onto PVDF blotting membrane (Amersham), through wet transfer for 1h 30 min at 200 mA in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membrane was blocked in 5% milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) and incubated overnight with primary antibodies diluted in 5% milk in TBST (anti-ELK1 (Abcam ab32106) 1:500, anti-TDP-43 (Abcam, ab104223) 1:2000, anti-tubulin (Sigma-Aldrich, MAB1637) 1:5000). After 1h-incubation with HRP-conjugated secondary antibodies diluted in 5% milk in TBST (anti-mouse HRP (BioRad, 1706516) 1:10000, anti-rabbit HRP (BioRad 1706515) 1:10000), the membrane was developed using Immobilon Classico HRP substrate (Sigma) and the Bio-Rad ChemiDoc system.