Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BTK autoinhibition analyzed by high-throughput swaps of SH2 domains

doi: 10.1073/pnas.2502688122

Figure Lengend Snippet: Screening αI kinase substitutions. The 118 αI kinase sequences are shown along with a multiple sequence alignment and fitness scores. The fitness scores are shown for the human BTK SH2 genetic background, the BMX-H SH2 genetic background, and the difference between these two backgrounds (BTK values subtracted from the BMX-H values). Error bars represent SEM and points represent the individual replicate values. For the difference values, error bars are determined using the SE propagation formula.

Article Snippet: The next day (day 2), cells were transfected with a mixture of transfer plasmid containing the SH2-BTK chimeras or the αI kinase chimeras (modified from pHIV-EGFP, Addgene #21373, 5 μg) and two packaging plasmids (pMDG.2, Addgene #12259, 1.25 μg and psPAX2, Addgene #12260, 3.75 μg) in 500 μL of opti-mem (Thermo Fisher) using Lipofectamine LTX (Thermo Fisher) according to the manufacturer’s instructions.

Techniques: Sequencing

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 1 Vps26a deficiency leads to the maintenance of stemness during ESC-mediated neurogenesis. a Effect of Vps26a deficiency on changes in alkaline phosphatase (AP) staining during neural differentiation (ND). Wild-type (WT; +/+) and Vps26a-/- (-/-) ESCs were differ- entiated in neurobasal medium (NBM) for the indicated time periods and subjected to AP staining. Cell clusters with differentiated morphologies are indicated by yellow dotted lines. Scale bar, 50 μm. b AP activities of WT and Vps26a-/- ESCs differentiated for 6 days indicated by scoring of the signal intensities of at least 60 colonies from three independent experiments. c The number of WT and Vps26a-/- cells during ND for 8 days. Error bars indicate the means ± standard deviation (SD; n = 3). ***P < 0.001 compared with WT cells each day during ND. d, e Effect of Vps26a deficiency on expression

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Staining, Standard Deviation, Expressing

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 2 Vps26a knockdown prolongs the maintenance of stemness under ND conditions in P19 ECCs. a, b The effect of Vps26a knockdown on the expression of ESC stemness genes were determined by semi-qPCR (a) and western blot (b) analyses of Oct3/4 and Nanog using shCTL- and shVps26a-ECCs. c Morphological changes of shCTL (shC)- and shVps26a (shV)-ECCs during retinoic acid-induced neurogenesis (RA-ND) for the indicated time periods. The yellow arrowheads indicate cells with differentiated morphologies. d–f Expression kinetics of ESC stemness and neuronal markers examined by semi-qPCR (d), qPCR (e), and western blotting (f)

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Knockdown, Expressing, Western Blot

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 4 Vps26a is required for increased ROS leading to ESC-mediated neurogenesis. a, b The effect of Vps26a deficiency (a) or knockdown (b) on ROS generation was determined by flow cytometry using WT and Vps26a-/- ESCs (a) and shCTL- and shVps26a-ECCs (#1, #10, and #14) (b) differentiated for 6 days and 48 h, respectively. Unstained cells (black lines) were used as a negative control. The data are representative of at least three independent experiments and presented as means ± SD (n = 3). ***P < 0.001. c WT and Vps26a-/- ESCs were differentiated in the presence or absence of 2.5 mM NAC for 6 days, and ROS levels were measured by flow cytometry. The data are

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Knockdown, Cytometry, Negative Control

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 5 Vps26a involves Nox expression during neurogenesis. a, b The effect of Vps26a deficiency and knockdown on the expression of the Nox family was determined by semi-qPCR (a) and qPCR (b) analyses using shCTL (shC)- and shVps26a (shV)-ECCs, differentiated for the indicated time periods. Error bars indicate the means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with shCTL-ECCs each day. c Over- view of Vps26a and Nox4 immunostaining of a sagittal section of WT

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Expressing, Knockdown, Immunostaining

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 6 Identification of the interaction between Vps26a and Nox4. a Serial sagittal sections of WT embryos were immunostained for Vps26a, Nox4, and Tubb3 and counterstained lightly with hematox- ylin at E10.5. da, dorsal aorta; l, lung; h, heart; nc, notochord; nl, neural lumen; nt, neural tube; som, somite. b Vps26a immunopreci- pitation followed by anti-Vps26a and -Nox4 immunoblots using lysates obtained from WT (+/+) and Vps26a-/- (-/-) ESCs during neural differentiation for 0 or 6 days. IgG was used as an immuno- precipitation control. c GST-tagged Vps26a was subjected to a pull- down assay with the lysates of HEK293 cells transfected with HA- Nox4C (C-terminal region, 249–574 aa)-expressing plasmid. Immu- noblot analysis with anti-HA antibody is shown at the top. Equal

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Western Blot, Immunoprecipitation, Control, Pull Down Assay, Transfection, Expressing, Plasmid Preparation

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 7 Nox4-generated ROS lead to a loss of stemness and subsequent neurogenesis from ESCs. a, b The effects of antioxidant and Nox inhibitor treatments on ROS generation (a) and ESC stemness tran- scription levels (b) were examined by flow cytometry using WT and Vps26a-/- ESCs differentiated in the presence or absence of 2.5 mM NAC or 10 μM DPI for 6 days. Error bars indicate the means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with no treat- ment. c Double-label immunocytochemical analysis of Vps26a (red),

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Generated, Cytometry

Journal: International Journal of Medical Sciences

Article Title: Parvovirus B19 Nonstructural Protein-Induced Damage of Cellular DNA and Resultant Apoptosis

doi:

Figure Lengend Snippet: PARP is active and necessary for efficient apoptosis in NS1-transfected cells. A. Immunoprecipitated GFP/NS1 protein was poly(ADP ribose)ylated, as shown by a band at 100 kd on the blot probed with anti-poly ADP ribose (PAR, left blot) antibodies. The blots were stripped and reprobed with anti-GFP (right), showing that PAR colocalizes with the GFP/NS1 fusion protein. GFP alone is not ribosylated, as evidenced by the lack of a PAR band corresponding to GFP at 37 kD. Blots shown are representative of three independent experiments. B. PARP activity is necessary for optimal NS1-induced apoptosis. Addition of the PARP inhibitor 5-aminoisoquinolinone (5AIQ) to GFP/NS1 expressing HepG2 cells reduced apoptosis by 57% ( p <0.003). Addition of 5-aminoisoquinolinone had no effect on the GFP transfected cells. N=3, error bars indicate the range of values.

Article Snippet: 25 μl of anti-GFP polyclonal antibody (Rockland, Gilbertsville, PA) were added and the mixture was allowed to bind for 14 hours at 4 o C in an end-over end rotator.

Techniques: Transfection, Immunoprecipitation, Activity Assay, Expressing