Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: ( A ) The mouse sterile alpha motif domain 14 ( Samd14) locus contains an E-box-GATA composite element (Samd14-Enh sequence highlighted) in intron 1 . The G1E wild type (WT) sequence and enhancer knockout G1E-derived cell clone sequence following TALEN directed enhancer knockout (G1E-ΔEnh) are shown. ( B ) Western blot of Samd14 and β-actin expression in G1E, WT clone #4 and G1E-ΔEnh clone. ( C ) Western blot of WT G1E cell lysates following pulldown with anti-Samd14 or anti-rabbit IgG control antibody. Input corresponds to 5% of immunoprecipitation (IP) lysate. ( D ) Western blot of G1E-ΔEnh cell lysates expressing empty vector (EV), hemagglutinin (HA)-Samd14 or HA-Samd14 Δ SAM following anti-HA pulldown. Blots were stained with anti-Samd14, anti-Capzα1, -Capzα2, -Capzβ, and β-actin antibodies. Input corresponds to 5% of IP lysate. ( E ) STRING plot depicting known interactions between capping protein (CP) complex subunits CAPZA1, CAPZA2, and CAPZB ( https://string-db.org/ ). ( F ) Western blot and semi-quantitative densitometry analysis of human CD34 + cell lysates at 4, 8, 12, 14, and 17 days of differentiation stained with anti-Capzβ and anti-Hsc70 antibodies. ( G ) Quantitation of Capzb , Capza1 , and Capza2 mRNA transcript levels in fluorescence-activated cell sorting (FACS) purified mouse bone marrow-derived hematopoietic cells. . Data from RNA-sequencing in . ( H ) Quantitation of CAPZB, CAPZA1, CAPZA2, and α-adducin (ADD1) protein copies per cell . Relative levels measured by quantitative mass spectrometry to determine absolute protein levels in human erythroid progenitors throughout differentiation stages. Prog1-Band3 - CD71 med GPA - , Prog2- Band3 - CD71 high GPA - , ProE- Band3 - CD71 high GPA low , Baso1- Band3 low CD71 high GPA med , Baso2- Band3 med CD71 high GPA high CD49d high , Poly- Band3 med CD71 high GPA high CD49d med , and Ortho-Band3 high CD71 med GPA high . MEP: megakaryocyte erythroid progenitor; EryA: Ter119 + CD71 + FSC high ; EryB: Ter119 + CD71 + FSC low . Figure 1—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Sterility, Sequencing, Knock-Out, Derivative Assay, Western Blot, Expressing, Control, Immunoprecipitation, Plasmid Preparation, Staining, Quantitation Assay, Fluorescence, FACS, Purification, RNA Sequencing, Mass Spectrometry

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: ( A ) Western blot of wild type (WT) G1E cell lysates after retroviral infection with control shRNA (sh Control ) or shRNA targeting Capzb mRNA (sh Capzb2 ) stained with anti-sterile alpha motif domain 14 (Samd14), anti-Capzα1, anti-Capzβ, or anti-Hsc70 antibodies. ( B ) Experimental layout. E14.5 mouse fetal liver progenitors are retrovirally infected with sh Control, sh Capzb1, or sh Capzb2 and cultured 3 days. R1–R5 flow cytometry gating using anti-CD71 and anti-Ter119 antibodies represents progressive stages of erythroid maturation. ( C ) Quantitation of Capzb mRNA in R1 (CD71 low Ter119 low ), R2 (CD71 high Ter119 low ), and R3 (CD71 high Ter119 high ) fetal liver progenitors from WT (N=3) mice in control (sh Control ) and following Capzb knockdown (sh Capzb1 and sh Capzb2 ). ( D ) Representative flow cytometry of E14.5 fetal liver progenitors expressing shControl , sh Capzb1, or sh Capzb2 using anti-CD71 and anti-Ter119 antibodies. ( E ) Quantitation of R1–R5 percentages in E14.5 fetal liver progenitors following retroviral infection with shControl , sh Capzb1, or sh Capzb2 and 3-day culture (N=3). Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed unpaired Student’s t test). Figure 2—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Western Blot, Retroviral, Infection, Control, shRNA, Staining, Sterility, Cell Culture, Flow Cytometry, Quantitation Assay, Knockdown, Expressing, Two Tailed Test

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: ( A ) Experimental layout of ex vivo spleen retroviral infection and cultures. ( B ) Relative mRNA expression of sterile alpha motif domain 14 ( Samd14) , Capzb, Capza1 , and Capza2 in control vs phenylhydrazine (PHZ)-treated CD71 + Kit + Ter119 - spleen cultures. ( C ) Quantitation of Capzb mRNA in fluorescence-activated cell sorting (FACS)-purified GFP + Kit + CD71 + cells from wild type (WT) mice following retroviral infection with shControl , sh Capzb2 after 2-day culture (left); Western blot of primary spleen cultures following Capzβ knockdown (sh Capzb2 ) or sh Control stained with anti-Capzβ and β-actin antibodies (right). ( D ) Quantitation of Samd14 mRNA in FACS-purified GFP + Kit + CD71 + cells from WT mice following retroviral infection with shControl , sh Capzb2 after 2-day culture. ( E ) Quantitation of infected R1–R5 cells in WT PHZ-treated cells following Capzb knockdown. ( F ) Representative images from Wright-Giemsa stained cells following Capzb knockdown (40 × magnification)( G ) Retrovirally-infected spleen progenitors were GFP-purified by FACS and grown for 2 days colony forming unit-erythroid (CFU-E) or 5 days burst forming unit-erythroid (BFU-E) and quantitated (N=9). ( H ) Representative flow cytometry scatter plot of membrane-impermeable DNA dye (Draq7) and anti-annexin V pacific blue (AnnV). Cells were first segregated on GFP + and Kit + gating. Live = Draq7 − AnnV − ; Early apoptotic (EA)=Draq7 − AnnV + ; Late apoptotic (LA)=Draq7 + AnnV + . ( I ) Quantitation of percent dead, LA and EA cells in the GFP + Kit + cells (left) and GFP + CD71 + Ter119 + (right). Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed unpaired Student’s t test). Figure 3—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Ex Vivo, Retroviral, Infection, Expressing, Sterility, Control, Quantitation Assay, Fluorescence, FACS, Purification, Western Blot, Knockdown, Staining, Flow Cytometry, Membrane, Two Tailed Test

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: (A ) Relative mRNA expression of Capzb, Capza1 , and Capza2 in control vs phenylhydrazine (PHZ)-treated spleen. ( B ) Western blot of control vs PHZ-treated spleens stained with anti-Capzβ and β-actin antibodies (left). Densitometry analysis of relative Capzβ expression in control vs PHZ-treated spleens (N=4) (right). ( C ) Quantitation of infected R1–R5 cells in sterile alpha motif domain 14-enhancer (Samd14-Enh -/- )PHZ-treated cells following Capzb knockdown (N=9). ( D ) Representative histogram of forward scatter area (FSC-A) in R2–R5 cells following sh Zb2 knockdown or sh Control (left); Quantitation of median FSC-A in sh Capzb2 vs sh Control (N=5) (right). ( E ) Quantitation of colony forming unit-erythroid (CFU-E) colonies in spleen precursors following Capzb knockdown (N=6). ( F ) Quantitation of percent dead (Draq7 + ) infected R1–R5 cells in wild type (WT) PHZ-treated spleen cells following Capzb knockdown (N=4). ( G ) Quantitation of percent dead (DAPI + ) in infected R1–R5 cells in untreated WT Lin - bone marrow cells following Capzb knockdown (N=4). ( H ) Quantitation of the percent of Ki67 + cells within CD71 low/med Kit + population following Capzb knockdown (N=4). Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed unpaired Student’s t test). Figure 3—figure supplement 1—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Expressing, Control, Western Blot, Staining, Quantitation Assay, Infection, Sterility, Knockdown, Two Tailed Test

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: ( A ) Schematic representation of Samd14 deletion construct location, protein sequence, and sequence alignment with known capping protein interacting (CPI) proteins. ( B ) Western blot of G1E-ΔEnh cell lysates expressing empty vectore (EV) or hemagglutinin (HA)-tagged Samd14, Samd14- Δ38–54, Samd14-Δ114–124, Samd14-Δ170–180, or Samd14-Δ272–295 constructs immunoprecipitation (IP) with anti-HA beads and stained with anti-HA, anti-Capzβ, anti-Capzα2, anti-Capzα1, or anti-β-actin antibodies. ( C ) Western blot of Lin- WT phenylhydrazine (PHZ)-treated spleen lysates immunoprecipitated (IPed) with anti-Samd14 or anti-rabbit IgG antibodies and stained with anti-Samd14 or anti-Capzβ antibodies. ( D ) Quantitation of colony forming unit-erythroid (CFU-E) (day 2 culture) and burst forming unit-erythroid (BFU-E) (day 5 culture) colonies in GFP + spleen progenitors expressing EV, HA-tagged Samd14, Samd14 ΔSAM, Samd14 ΔCPB, and Samd14 ΔCPBΔSAM constructs (N=6). Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed unpaired Student’s t test). Figure 4—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Construct, Sequencing, Western Blot, Expressing, Immunoprecipitation, Staining, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: (A ) Western blot of Lin - wild type (WT) spleen lysates obtained from mice treated with either PBS (control) or phenylhydrazine (PHZ) (100 mg/kg) immunoprecipitated (IPed) with anti-sterile alpha motif domain 14 (Samd14) or anti-rabbit IgG antibodies and stained with anti-Samd14, or anti-Capzβ antibodies (left). Densitometry analysis of the relative CapZβ co-IPed with Samd14 in PBS (control) vs PHZ (right). ( B ) Western blot of R2 (CD71 + Ter119 - ) and R3 (CD71 + Ter119 + ) cells sorted from WT PHZ-treated spleen lysates IPed with anti-Samd14 or anti-rabbit IgG antibodies and stained with anti-Samd14 or anti-Capzβ antibodies. ( C ) Western blot of Lin-WT PHZ-treated spleen cells treated with +/-stem cell factor (SCF) (10 ng/ml) for 5 min and IPed with anti-Samd14 or anti-rabbit IgG antibodies and stained with anti-Samd14 or anti-Capzβ antibodies (left). Densitometry analysis of the relative CapZ co-IPed with Samd14 in the presence or absence of SCF (right). Figure 4—figure supplement 1—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Western Blot, Control, Immunoprecipitation, Sterility, Staining

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet: ( A ) Sequence alignment of the capping protein binding (CPB) domain of Samd14 among vertebrate species. Aligned using Clustal Omega and visualized with JalView. Yellow bars in conservation plot represents evolutionary conservation. ( B ) Sequence alignment of the capping protein interaction (CPI) motif in other proteins with Samd14 CPB domain. Yellow color indicates conserved residues among the different CPI motifs. ( C ) Schematic representation of chimeric and mutated CPB domain Samd14 constructs. ( D ) Western blot of GFP-sorted G1E-ΔEnh cells expressing empty vector (EV), hemagglutinin (HA)-Samd14, Samd14-∆CPB, Samd14(S14)-Ckip1-CPI, or Samd14-R43/45 A stained with anti-HA. HA and β-actin ( E ) Western blot analysis of Capzα1, Capzα2, and Capzβ co-immunoprecipitated (IPed) following pull-down of EV, HA-tagged Samd14, Samd14-∆CPB, Samd14(S14)-Ckip1-CPI and Samd14-R43/45 A with anti-HA agarose beads from retrovirally infected G1E-ΔEnh cells. ( F ) Densitometry analysis of the relative CapZ co-IPed with either HA-Samd14 or HA-S14-R43/45 R. ( G ) Quantitation of GFP + colony forming unit-erythroid (CFU-E) (N=9) and burst forming unit-erythroid (BFU-E) (N=17) colonies formed in spleen progenitors retrovirally infected with EV, HA-tagged Samd14, Samd14-∆CPB, Samd14(S14)-Ckip1-CPI, and Samd14-R43/45 A. Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed unpaired Student’s t test). Figure 6—source data 1. Source Western blot images for .

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Sequencing, Protein Binding, Construct, Western Blot, Expressing, Plasmid Preparation, Staining, Immunoprecipitation, Infection, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

doi: 10.7554/eLife.76497

Figure Lengend Snippet:

Article Snippet: Antibody , anti-capzβ (mouse monoclonal) , Santa Cruz Biotechnology , Cat.: #sc-136502; RRID: AB_10610091 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Software