Cofilin

Mouse: Sox2 (Santa Cruz, sc-365823, 1:500), Hopx (Santa Cruz, sc-398703, 1:250), Cofilin (Santa Cruz, sc-376476, 1:100), Cdc42 (Santa Cruz, sc-8401, 1:200) Cux1 (Abcam, AB54583, 1:500), pVim (Fisher, 5045934, 1:500), pHistone H3 (abcam, ab14955, 1:500), Cryab (ab13496, 1:500), Rabbit: Hopx (Proteintech, 11419–1-AP, 1:500), pS6 (Cell Signaling, 2211S, 1:500), p4EBP1 (Cell Signaling Technology, 2855S, 1:500), DEPDC5 (Sigma, HPA054969, 1:500), Arp2 (Proteintech, 10922–1-AP, 1:100), Cleaved Caspase-3 (9661S, 1:200). Rat: Ctip2 (Abcam, ab18465, 1:500), BrdU (Abcam, ab6326, 1:500). Sheep: Eomes (R and D, AF6166, 1:200), Goat: Sox2 (Santa Cruz, sc-17320, 1:250), Guinea pig: NeuN (Millipore, ABN90, 1:500), Chicken: Gfp (Aves, GFP-1020, 1:500).

Osteoclast cultures were fixed with 4% paraformaldehyde in PBS and incubated for 15 min at room temperature. The cells were subsequently permeabilized with 0.1% Triton X-100 or 0.01% Tween-20 in PBS on ice for 5 min. To prevent non-specific binding, samples were preincubated in 1% BSA–PBS for 30 min. The following fluorochrome conjugates were used with 1:100 dilution in 1% BSA in PBS: ATTO 647-Phalloidin (Sigma-Aldrich, 65906), or Alexa Fluor^® 488 Phalloidin (Molecular Probes, Eugene, OR, A12379). Nuclei were visualized with a Hoechst 33342 (Molecular Probes, H3570) with a 1:10 000 dilution. To study the potential binding partners of actin, the cells were double labeled for actin and c-Src (Cell Signaling, 2109), cortactin (Santa Cruz, 55579), anti-arp2 antibody (Abcam, ab47657), or cofilin (Santa Cruz, 53934). For secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (Abcam, Cambridge, UK) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, OR, USA) antibodies were used. After immunolabeling, the coverslips were mounted with Mowiol (Sigma-Aldrich).

miR-29b/a plasmid was purchased from Addgene (Cambridge, MA, USA). Plasmids for shB7-H3 were purchased from Origene (Rockville, MD, USA). B7-H3 overexpression plasmid (B7-H3 OE) was purchased from Sino Biological Inc. (Wayne, PA, USA). A pCDNA3-scrambled vector was used as a control. Antibodies for B7-H3, c-Myc, p-STAT1, STAT1, IGF-1R, Cofilin, N-cadherin, CDK-6, Caspase 3, and Actin were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA). shMYC siRNA was also purchased from Santa Cruz. HRP conjugated secondary antibodies were purchased from Novus Biologicals (Littleton, CO, USA). The MYC inhibitor, JQ1, was purchased from Cayman Chemicals (Ann Arbor, MI, USA). All-trans retinoic acid (ATRA) was purchased from Sigma Aldrich (St. Louis, MO, USA).

The drugs used for screening in this study are listed in Supplementary Table 1.
Reversine, SP600125, ZM 447439, BI 2536, STLC, Dimethylenastron, SB203508, RO 3306, Cdk1 Inhibitor III, Roscovitine, NSC 95397, IPA3, Y-27632, ITX-3, Nocodazole, Paclitaxel, Blebbistatin, Cytochalasin B, MG 132 and Velcade were purchased from Sigma–Aldrich (St. Louis, MO, USA). AZ 3146 and Mps-BAY2a were obtained from Tocris (Bristol, United Kingdom). MLN8237 (Alisertib), AZD1152-HQPA (Baraserib) and SB743921 were purchased from Selleckchem. GSK923295 is from MedChem express.
Monoclonal antibodies against α-tubulin, CDK1, AURKA, PLK1, KIF11 (Sigma-Aldrich); CCNB1, CCNE1, TP53, CDKN1A, JNK1, JNK2 (Cell signaling); EB1, COFILIN (Santa Cruz), TTK, BUBR1 (Abcam). Polyclonal antibodies against PRC1 (Santa Cruz), Tpx2 [69 (link)]; Kif23 [70 (link)] were used.

Y27632 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Z-VAD-fmk was purchased from EMD Biosciences (La Jolla, CA). Antibodies against Akt, cytochrome c, cofilin, actin, cleaved ROCK1, PTEN, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); cleaved caspase-3, cleaved caspase-9, phospho-Akt (Ser473), phosphor-cofilin (Ser3), PI3K, phospho-PI3K, RhoA, and Cox IV were purchased from Cell Signaling Technology (Beverly, MA); ROCK1 was obtained from Abcam (Burlingame, CA); PARP was obtained from Biomol (Plymouth Meeting, PA).

Antibodies: Arc C7 mouse monoclonal (1:200, sc-17839), Arc H300 rabbit polyclonal (1:200, sc-15325), Cofilin (1:500, sc-32158), Drebrin A (1:200, #sc-374269), Dynamin 2 (1:1000, sc-6400), GAPDH (1:5000, sc-32233), Histone 1 (1:500, sc-10806), rabbit polyclonal SUMO1 (1:1000, sc-9060), mouse monoclonal SUMO1 (1:1000, sc-5308), SUMO2/3 (1:1000, sc-32873), normal mouse IgG and rabbit IgG were from Santa Cruz Biotechnology. Arc Synaptic Systems (1:1000, 156003) β-actin (1:5000 Sigma, #F3022), Cofilin (1:500, Cell signaling #5175), Drebrin A (1:500, Cell signaling #12243S), CaMKIIα (1:500, Chemicon, #MAB8699), CaMKIIβ (Invitrogen #139800), His6-tag (1:1000, Millipore #5531), PSD-95 (1:1000, Thermo scientific #MA1-045), Vimentin (1:1000, Sigma #V5225).
Recombinant TrkB-Fc (stock 100 μg/ml, #688-TK) and control human IgG-Fc (100 μg/ml, #110-HG) were obtained from R&D Systems and diluted in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. The cDNA encoding Arc was a gift from Dr. Joseph Dynes, University of California, Irvine, USA. Expression constructs for His6-tagged SUMO1, 2 and 3 were kindly provided by Dr. Ronald Hay, University of Dundee, UK.