Journal: Cell Death & Disease

Article Title: MEIS2 is essential for neuroblastoma cell survival and proliferation by transcriptional control of M-phase progression

doi: 10.1038/cddis.2014.370

Figure Lengend Snippet: MEIS2 is essential for neuroblastoma cell survival. ( a ) Immunoblotting of MEIS2 in a panel of 13 human neuroblastoma cell lines. β -Actin levels are shown as a loading control. ( b and c ) qRT-PCR ( b ) and immunoblot ( c ) analyses of MEIS2 expression in BE(2)-C cells infected with lentiviruses expressing either shGFP or shMEIS2. Error bars ( b ) represent S.D. ( n =3). GAPDH ( c ) serves as a loading control. ( d ) Micrographs of BE(2)-C cells infected for 3 days with lentiviruses expressing either shGFP or shMEIS2. ( e ) Trypan blue exclusion assay of viable BE(2)-C cells infected for 4 days with lentiviruses expressing either shGFP or shMEIS2

Article Snippet: Immunoblotting was conducted according to standard procedures using the following primary antibodies: rabbit anti-BCL2 (sc-783, 1 : 200), rabbit anti-caspase 3 (sc-7148, 1 : 200), rabbit anti-Flag (F7425, 1 : 1000; Sigma-Aldrich), rabbit anti-FOXM1 (sc-502, 1 : 100), rabbit anti-GAPDH (sc-25778, 1 : 3000), mouse anti-MEIS2 (sc-81986, 1 : 400), rabbit anti- β -actin (600-401-886, 1 : 2000; Rockland Immunochemicals, Gilbertsville, PA, USA), and mouse anti- α -tubulin (B-5-1-2, 1 : 5000; Sigma-Aldrich).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Infection, Trypan Blue Exclusion Assay

Journal: Cell Death & Disease

Article Title: MEIS2 is essential for neuroblastoma cell survival and proliferation by transcriptional control of M-phase progression

doi: 10.1038/cddis.2014.370

Figure Lengend Snippet: MEIS2 targets the MuvB-BMYB-FOXM1 complex for transcriptional control of M-phase progression. ( a ) qRT-PCR analysis of mRNA expression of MEIS2, FOXM1, BMYB, and RBBP4 in BE(2)-C cells infected with lentiviruses expressing either shGFP or shMEIS2. ( b ) Immunoblotting of MEIS2 and FOXM1 in BE(2)-C cells infected with lentiviruses expressing either shGFP or shMEIS2. β -Actin levels are shown as a loading control. MEIS2 and FOXM1 levels were quantified against β -actin. ( c ) GSEA showing marked downregulation of the FOXM1 pathway genes in BE(2)-C cells with MEIS2 depletion. ( d ) ChIP-qPCR analysis showing MEIS2 binding to the FOXM1 promoter region (−312 to −158) containing a consensus TGIF/MEIS2-binding sequence TGTCA. Error bars, S.D. ( n =3). ( e ) Immunoblotting of FOXM1 in BE(2)-C and SK-N-DZ cells with or without FOXM1 knockdown. β -Actin levels are shown as a loading control. ( f ) qRT-PCR analysis of mRNA expression of key FOXM1 target genes in BE(2)-C and SK-N-DZ cells with or without FOXM1 knockdown. ( g ) Growth assay of BE(2)-C and SK-N-DZ cells with or without FOXM1 knockdown. Error bars, S.D. ( n =3). ** P <0.001. Data ( d and g ) were analyzed with two-tailed Student's t -test

Article Snippet: Immunoblotting was conducted according to standard procedures using the following primary antibodies: rabbit anti-BCL2 (sc-783, 1 : 200), rabbit anti-caspase 3 (sc-7148, 1 : 200), rabbit anti-Flag (F7425, 1 : 1000; Sigma-Aldrich), rabbit anti-FOXM1 (sc-502, 1 : 100), rabbit anti-GAPDH (sc-25778, 1 : 3000), mouse anti-MEIS2 (sc-81986, 1 : 400), rabbit anti- β -actin (600-401-886, 1 : 2000; Rockland Immunochemicals, Gilbertsville, PA, USA), and mouse anti- α -tubulin (B-5-1-2, 1 : 5000; Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot, Binding Assay, Sequencing, Growth Assay, Two Tailed Test

Journal: Central European Journal of Immunology

Article Title: Experimental immunology Potential role of RING finger protein 166 (RNF166), a member of an ubiquitin ligase subfamily, involved in regulation of T cell activation

doi: 10.5114/ceji.2013.34353

Figure Lengend Snippet: Fig. 1. Endogenous expression analysis of RNF166 isoform 1 in T lymphoid cells. A) Amino acid alignments of three isoforms of human RNF166 protein, encoded by transcript variant 1 (NM_178841.3), transcript variant 2 (NM_001171815.1) and tran- script variant 3 (NM_001171816.1) respectively. Alignments were performed using DNAMAN software (Lynnon, Quebec, Canada). Identical residues are highlighted in black. Block symbols indicate the domains of RNF166. Underlines indicate the co-owned recognized sites of the polyclonal antibody. B) Isoform 1 (26.1 kDa) of RNF166 protein was detected both in primary T cells and Jurkat T cells by western blot analysis, confirming that RNF166 can be endogenously expressed in T lymphoid cells (lane: 1. Jurkat T cells transfected with pEGFP-RNF166; 2. untransfected Jurkat T cells; 3. untransfected primary T cells); anti- GFP and anti-β-actin antibodies were employed as controls

Article Snippet: The membranes were incubated overnight at 4°C with the indicated primary antibody: rabbit polyclonal anti-GFP antibody (eBioscience, USA); rabbit polyclonal anti-human RNF166 antibody (Abcam, USA) and rabbit polyclonal anti-human beta actin antibody (AVIVASystems Biology, USA), followed by 5 min washes in TBS-T for three times and incubation with HRPlabeled secondary antibody (Zhongshan Goldenbridge, China) in TBS-T for 1 hour at RT.

Techniques: Expressing, Variant Assay, Software, Blocking Assay, Western Blot, Transfection