Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: NCAM is dynamically expressed in NPCs during cortical development. (A and B) Coronal sections of mouse cortices from indicated embryonic stages were coimmunostained for NCAM and Sox2 (A) or Tuj1 (B). Scale bars, 50 µm. (C) Percentages of NCAM + immunoreactivity in each layer. (D) Average immunofluorescence density of NCAM in each layer. n = 9 brain slices from three mice. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with Bonferroni corrections (IZ, CP, and MZ in C and MZ in D), Dunnett’s T3 correction (VZ/SVZ in C), and Kruskal-Wallis test with Dunn-Bonferroni correction (VZ/SVZ, IZ, and CP in D). PP, preplate.

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, shRNA

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Cell Culture, Transfection, Incubation, shRNA, Plasmid Preparation, Expressing

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Western Blot, Cell Culture, Mutagenesis, Expressing, Lysis, Transduction, Staining, Incubation, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

doi: 10.1083/jcb.201902164

Figure Lengend Snippet: The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.

Article Snippet: The following antibodies were used for immunofluorescence analysis: goat anti-Sox2 (1:150, sc-17320, RRID: AB_2286684; Santa Cruz Biotechnology), mouse anti-BrdU (1:300, MMS-139S, RRID: AB_10719257; Covance), mouse anti–βIII-tubulin (1:500, Tuj1, T5076, RRID: AB_532291; Sigma-Aldrich), mouse anti-profilin2 (1:100, 60094-2-Ig, RRID: AB_2163215; Proteintech), rabbit anti-NCAM (1:300, ANR-041, RRID: AB_2756690; Alomone Labs), rabbit anti-NCAM (1:200, 701379, RRID: AB_2532477; Thermo Fisher Scientific), rabbit anti-Pax6 (1:300, PRB-278P, RRID: AB_291612; Covance), rabbit anti-Tbr1 (1:300, ab3190, RRID: AB_2238610; Abcam), rabbit anti-Tbr2 (1:300, ab23345, RRID: AB_778267; Abcam), rabbit anti-Olig2 (1:300, ab109186, RRID: AB_10861310; Abcam), rabbit anti-GFAP (1:500, MAB360, RRID: AB_11212597; EMD Millipore), rabbit anti-PH3 (1:300, 06-570, RRID: AB_310177; EMD Millipore), rabbit anti-Ki67 (1:100, PA5-19462, RRID: AB_10981523; Thermo Fisher Scientific), rabbit anti-Ctip2 (1:300, ab28448, RRID: AB_1140055; Abcam), rabbit anti-Cux1 (1:100, sc-13024, RRID: AB_2261231; Santa Cruz Biotechnology), rabbit anti-BLBP (1:100, ab32423, RRID: AB_880078; Abcam), mouse anti-A2B5 (1:300, MA1-90445, RRID: AB_1954783; Thermo Fisher Scientific), and rabbit anti–cleaved caspase3 (1:300, 9661, RRID: AB_2341188; Cell Signaling Technology).

Techniques: Expressing

Journal: Journal of Thoracic Disease

Article Title: The value of cell block based on fine needle aspiration for lung cancer diagnosis

doi: 10.21037/jtd.2017.07.91

Figure Lengend Snippet: Immunoreactivity for TTF1 and CD56 in small cell lung carcinoma. (A) Poorly differentiated small cell lung carcinoma in TCT (H & E, ×400); (B) SCLC in cell blocks (H & E, ×100); (C) tumor cells are positive for TTF1 (×400); (D) tumor cells are positive for CD56 (×400). SCLC, small cell lung carcinoma; TCT, ThinPrep cytology test.

Article Snippet: Antibodies to the following molecules were used: P40 (P40, 1:500, OriGene), TTF-1 (8G7G3/1, 1:200, Dako), SYN (DAK-SYNAP, 1:100, Dako), CD56 {123C3[5], 1:50, Dako}, CgA (DAK-A3, 1:100, Dako), LCA (2B11 + PD7/26, 1:100, Dako), CK5/6 (D5/16B4, 1:100, Dako) and Ki-67 (MIB-1, 1:100, Dako).

Techniques:

Journal: Biomolecules

Article Title: IL-18BP Improves Early Graft Function and Survival in Lewis–Brown Norway Rat Orthotopic Liver Transplantation Model

doi: 10.3390/biom12121801

Figure Lengend Snippet: IL-18BP overexpression reduced CD56 expression after LEW-BN OLTx. ( A ) Representative images of IHC staining for CD56 protein in livers of different groups. Scale bar, 50 μm, 25 μm. ( B ) The cumulative frequency of tissue-infiltrating CD56 + NK cells detected by IPP 6.0 in each group; error bars represent means ± SEM ( n = 3 per group); * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibody was rabbit anti-CD56 monoclonal antibody (14255-1-AP, proteintech, Wuhan, China) (1:2000 dilution; 120 min at room temperature).

Techniques: Over Expression, Expressing, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators

doi: 10.1186/s12967-022-03387-7

Figure Lengend Snippet: Identification and characterization of Immune cell subtypes in SIMC and PBMC. a the proportions of immune cells calculated by CIBERSORT, N: controls, P: pemphigus patients. b Barplot of CIBERSORT scores of SIMC in pemphigus and control skin samples. c the heatmap of deferentially expressed chemokine and chemokine receptor in SIMC between pemphigus patients and healthy controls. d Correlation heatmap of different types of immune cells. e Immunohistochemistry staining of CD56 in skin, CD56: NK cell marker. f GSEA enrichment plots of GSE37532, and GSE24574. Normalized enrichment score (NES) indicated the analysis results across gene sets. g GSEA analysis result of SIMC database

Article Snippet: The sections were blocked with 10% normal goat serum in Tris–HCl-buffered saline or horse serum in PBS for 1 h and then incubated with primary anti-NCAM1/CD56 antibodies(Clone number: EP2567Y; Abcam, Waltham, USA) at a concentration of 1:200 for 1 h at room temperature or overnight at 4℃.

Techniques: Immunohistochemistry, Staining, Marker

Journal: bioRxiv

Article Title: Upregulation of FasII underlies synergistic neuropathological and behavioral defects in a Drosophila model of myotonic dystrophy

doi: 10.1101/2024.05.26.595976

Figure Lengend Snippet: a, b Representative semi-quantitative RT-PCR of fasII in Drosophila larval (a) CNS and (b) BWM. c Quantification of a . n = 3 for both genotypes. d Quantification of b . n = 3 for both genotypes. e, f Representative slot blots of FasII in adult Drosophila (e) head and (f) muscle. g Quantification of e . n = 3 for both genotypes. h Quantification of f . n = 3 for both genotypes. i, j Representative slot blots of NCAM1 in mouse (i) head and (j) muscle. k Quantification of i . n = 3 for both genotypes. l Quantification of j . n = 3 for both genotypes. m Quantified slot blot data of NCAM1 in human frontal cortex. n = 5 for control. n = 10 for DM1 patients. n Quantitative dot blot data of NCAM1 in human transdifferentiated myotube. n = 4 for both control and DM1 patients. Histograms depicts mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were blocked in 5% Blotto (Santa Cruz Biotech, sc-2324) diluted in 1X TBS-T (10 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween 20) for 1 hour at room temperature, and incubated overnight at 4°C with primary anti-NCAM1 antibody (GeneTex, GTX111684) diluted 1:10,000 in 5% Blotto.

Techniques: Quantitative RT-PCR, Dot Blot, Control, Quantitative Dot Blot

Journal: The International journal of biological markers

Article Title: Effect of secretory DKK3 on circulating CD56 bright natural killer cells in patients with liver cancer.

doi: 10.1177/03936155231169796

Figure Lengend Snippet: Figure 2. The roles of recombinant human DKK3 in the cytotoxicity and differentiation of CD56bright NK cells in vitro. (a) CD56, phosphorylation and total GSK-3β, and NF-κB p65 of CD56bright NK cells was detected using western blot. (b) NKG2D expression of CD56bright NK cells was detected using flow cytometry. (c) The cytotoxicity of CD56bright NK cells was determined using LDH release assay. LDH: lactate dehydrogenase; NK: natural killer.

Article Snippet: The nitrocellulose (NC) membranes were incubated with primary antibody for CD56 (sc-7326), p-GSK-3β (sc-81496), GSK-3β (sc-7291), GAPDH (sc-47724) (Santa Cruz, CA, USA), p-NF-κB p65 (ab239882), and NF-κB p65 (ab32536) (Abcam, Shanghai, China) overnight at 4°C.

Techniques: Recombinant, In Vitro, Phospho-proteomics, Western Blot, Expressing, Cytometry, Lactate Dehydrogenase Assay