American Society of Hematology

Figure 4.

$CRLF3 interacts with the Hippo pathway. (A) Western blot of sucrose gradient centrifugation fractionated human platelets probed with antibodies against α-tubulin, β-actin, thrombospondin (THBS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and CRLF3. Fractions 1 to 5 represent cytoskeletal proteins (enriched for α-tubulin and β-actin), whereas fractions 7 and 8 represent granular proteins (enriched for THBS-1). (B) Representative flow cytometric plots of CRLF3 TAP-tagged and control forward programmed iPSC MKs stained with CD41a and CD42a. (C) Western blot of CRLF3 TAP-tagged and control iPSCs and iPSC MKs probed with antibodies against FLAG (green) and GAPDH (red). (D) CRLF3 TAP-tagged iPSC MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 24 hours to induce proplatelet formation. Samples were fixed, stained with α-tubulin (red), FLAG (green), and DAPI (blue), and imaged by fluorescence microscopy. Subcellular distribution of α-tubulin and FLAG staining in round and proplatelet-forming CRLF3 TAP iPSC MKs was determined across a section of the MKs along the indicated arrow using ImageJ. Scale bars are 50 μm. (E-F) CRLF3 TAP (E) and control (F) iPSC MKs were lysed and immunoprecipitated (IP) with antibodies against FLAG, MOB1, and immunoglobulin G (IgG). Precipitated lysates were then probed for STK38, MOB1, and FLAG by western blot. (G) In vitro cultured MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 5 hours to induce proplatelet formation. Samples were fixed, stained for MOB1 (green), α-tubulin (red), and DAPI (blue), and imaged by fluorescence microscopy. Images are representative for Crlf3−/− and control (WT) proplatelet-forming MKs. (H) Western blot of in vitro cultured MKs probed with antibodies against pMOB1, MOB1, and GAPDH (left panel; n = 8 MOB1/GAPDH and 4 pMOB1/GAPDH) and pSTK38, STK38 and GAPDH (right panel; n = 3 STK38/GAPDH, 3 Crlf3−/−, and 4 WT pSTK38/GAPDH). (I) Three-dimensional structure of CRLF3 (residue 174 to end) solved by experimental phasing. Domains are labeled. Molecular graphics prepared using PyMOL. Data represent means ± standard deviations. Unpaired 2-tailed Student t test. *P < .05. FITC, fluorescein isothiocyanate; FN3, fibronectin type 3.$

CRLF3 interacts with the Hippo pathway. (A) Western blot of sucrose gradient centrifugation fractionated human platelets probed with antibodies against α-tubulin, β-actin, thrombospondin (THBS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and CRLF3. Fractions 1 to 5 represent cytoskeletal proteins (enriched for α-tubulin and β-actin), whereas fractions 7 and 8 represent granular proteins (enriched for THBS-1). (B) Representative flow cytometric plots of CRLF3 TAP-tagged and control forward programmed iPSC MKs stained with CD41a and CD42a. (C) Western blot of CRLF3 TAP-tagged and control iPSCs and iPSC MKs probed with antibodies against FLAG (green) and GAPDH (red). (D) CRLF3 TAP-tagged iPSC MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 24 hours to induce proplatelet formation. Samples were fixed, stained with α-tubulin (red), FLAG (green), and DAPI (blue), and imaged by fluorescence microscopy. Subcellular distribution of α-tubulin and FLAG staining in round and proplatelet-forming CRLF3 TAP iPSC MKs was determined across a section of the MKs along the indicated arrow using ImageJ. Scale bars are 50 μm. (E-F) CRLF3 TAP (E) and control (F) iPSC MKs were lysed and immunoprecipitated (IP) with antibodies against FLAG, MOB1, and immunoglobulin G (IgG). Precipitated lysates were then probed for STK38, MOB1, and FLAG by western blot. (G) In vitro cultured MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 5 hours to induce proplatelet formation. Samples were fixed, stained for MOB1 (green), α-tubulin (red), and DAPI (blue), and imaged by fluorescence microscopy. Images are representative for Crlf3^−/− and control (WT) proplatelet-forming MKs. (H) Western blot of in vitro cultured MKs probed with antibodies against pMOB1, MOB1, and GAPDH (left panel; n = 8 MOB1/GAPDH and 4 pMOB1/GAPDH) and pSTK38, STK38 and GAPDH (right panel; n = 3 STK38/GAPDH, 3 Crlf3^−/−, and 4 WT pSTK38/GAPDH). (I) Three-dimensional structure of CRLF3 (residue 174 to end) solved by experimental phasing. Domains are labeled. Molecular graphics prepared using PyMOL. Data represent means ± standard deviations. Unpaired 2-tailed Student t test. *P < .05. FITC, fluorescein isothiocyanate; FN3, fibronectin type 3.

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