Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: WBP-1 virus using mouse ACE2 to gain access to cells was determined by immunofluorescence. Hela cells with transfection of empty vector (Flag), hACE2, or mACE2 plasmids were infected with WT Wuhan-Hu-1 or mouse-adapted WBP-1. ACE2 expression was detcted using rabbit anti-ACE2 IgG followed by CoraLite594-conjugated goat anti-rabbit IgG (H+L). SARS-CoV-2 nucleocapsid expression was detected using mouse anti-NP IgG followed by CoraLite488-conjugated Affinipure goat anti-mouse IgG(H+L). Nuclei were stained with DAPI. Scale bars, 200 μm.

Article Snippet: Proteins used in the BLI experiment (hACE2, mACE2, WT-RBD, Q493K-RBD, Q498H-RBD, Q493K/Q498H-RBD) were purchased from Sino Biological.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Infection, Expressing, Staining

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: Mouse-adapted WBP-1 virus RBD enhance interactive affinities with mACE2. (a-d) Modelling of SARS-CoV-2 RBD–ACE2 interface. (a) Wuhan-Hu-1 RBD interacts with of human ACE2. (b) WBP-1 RBD (Q493K/Q498H-RBD) interacts human ACE2. (c) Modelling of Wuhan-Hu-1 RBD and mouse ACE2. (d) Modelling of WBP-1 RBD (Q493K/Q498H-RBD) shows interaction with mouse ACE2. (e-l) The binding affinities of the RBD of WBP-1 and mACE2 or hACE2 were determined through BLI experiments. The sensors were dipped in mACE2-hFC and functionalized sensorgrams captured upon incubation of Q493K-RBD (e), Q498H-RBD (f), Q493K/Q498H-RBD (g), and WT-RBD (h) at 6.25 (black), 12.5 (blue), 25 (green), and 50 nM (red). The sensors were dipped in hACE2-hFC and functionalized sensorgrams captured upon incubation of Q493K-RBD (i), Q498H-RBD (j), Q493K/Q498H-RBD (k), and WT-RBD (l) at 50 (black), 100 (blue), 200 (green), and 400 nM (red).

Article Snippet: Proteins used in the BLI experiment (hACE2, mACE2, WT-RBD, Q493K-RBD, Q498H-RBD, Q493K/Q498H-RBD) were purchased from Sino Biological.

Techniques: Binding Assay, Incubation

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-WT, but not ALK2-R206H, forms a significant amount of homomeric complexes. COS7 cells were co-transfected with expression vectors encoding myc-tagged ALK2-WT or ALK2-R206H alone or together with their HA-tagged counterpart (or empty vector). Where shown, the HA-tagged receptor was immobilized by IgG-crosslinking as in , following the schematic description in . Thus, the HA-tagged receptor was patched and crosslinked by IgGs, and the myc-tagged version of the same receptor was labeled by Fab’ fragments. The lateral mobility of the Fab’-labeled myc-tagged receptor was measured by FRAP. Where indicated, ActA (4 nM) was added where indicated as in . ( A , C ) Average R f values; ( B , D ) average D values. Bars are mean ± SEM; the number of measurements (each conducted on a different cell) is shown within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). A similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Labeling

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B forms stable heteromeric complexes with either ALK2-WT or ALK2-R206H. COS7 cells were co-transfected with expression vectors encoding myc-ACVR2B alone or together with HA-tagged ALK2 (WT or R206H). Where indicated, HA-ALK2 was immobilized by IgG-crosslinking as in , as shown schematically in . The HA-tagged receptor (ALK2-WT or ALK2-R206H) was patched and crosslinked by IgGs, and the co-expressed myc-ACVR2B was labeled exclusively by monovalent Fab’; the lateral mobility of Fab’-labeled myc-ACVR2B was measured by FRAP, without or with ActA (4 nM). ( A ) Quantification of the cell surface levels of myc-ACVR2B alone or co-expressed with HA-tagged ALK2 variants. Myc-ACVR2B cell surface receptors were labeled at 4 °C by a saturating concentration (40 μg/mL) of murine Fab’ αmyc, followed by 40 μg/mL Alexa 546-Fab’ GαM, and fixed (4% paraformaldehyde). This protocol enables the measurement of the levels of the tagged receptors at the plasma membrane under identical conditions (same laser excitation line and intensity, same microscope filters, same settings of the photomultiplier tube) [ , ]. The surface levels of the receptors were quantified by measuring the fluorescence intensity from a point-confocal spot by the FRAP apparatus under non-bleaching conditions as described) [ , ]. Data are mean ± SEM of 30 measurements under each condition. No significant differences were observed between the values under the different conditions. ( B , D ) Average R f values; ( C , E ) average D values. Bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (**, p < 4 × 10 −3 ; ***, p < 7 × 10 −4 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, Expressing, Labeling, Concentration Assay, Membrane, Microscopy, Fluorescence

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2A forms heteromeric complexes with ALK2-WT to a much higher degree than with ALK2-R206H. Experiments were as in , following the scheme depicted in , except that Fab’-labeled myc-ACVR2A replaced myc-ACVR2B. Where indicated, HA-ALK2-WT or HA-ALK2-R206H were immobilized by IgG-crosslinking, as in . The lateral mobility of Fab’-labeled myc-ACVR2A was measured by FRAP, without or with ActA (4 nM; see ). ( A ) Quantification of the cell surface levels of myc-ACVR2A alone or co-expressed with HA-tagged ALK2 variants. The experiment was conducted exactly as described in A. Data are mean ± SEM of 30 measurements under each condition. No significant differences were observed between the surface levels of myc-ACVR2A alone or co-expressed with HA-tagged ALK2 (WT or R206H). ( B , D ) Average R f values; ( C , E ) average D values. Bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; ***, p < 8 × 10 −4 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Labeling

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B, but not ACVR2A, enhances ALK2-WT homomeric interactions in the presence of ActA. Patch/FRAP studies were conducted on COS7 cells expressing myc-ALK2-WT alone or together with HA-ALK2-WT. Where indicated, untagged ACVR2A, ACVR2B, or ACVR2B-KD were co-expressed. CL marks IgG-mediated crosslinking of HA-ALK2-WT, performed as in A,B. The schematics of the experimental design are depicted in ; HA-ALK2-WT was patched and crosslinked by IgGs, while myc-ALK2-WT was labeled by Fab’ fragments. Where indicated, an untagged ACVR2 receptor variant was co-expressed as a third receptor to measure its effect. The lateral mobility of the Fab’-labeled myc-ALK2-WT was measured by FRAP. ( A ) Control experiments showing that the cell surface levels of myc-ALK2-WT are not significantly affected by co-expression with untagged ACVR2A or ACVR2B. The experiment was conducted as described in A, except that the surface levels of myc-ALK2-WT were measured by the point confocal method (see A). Data are mean ± SEM of 30 measurements in each case. No significant differences were observed between the surface levels of myc-ALK2-WT alone or co-expressed with untagged ACVR2A or ACVR2B (one-way ANOVA and Bonferroni post hoc test). ( B ) Average R f values; ( C ) average D values. Bars depict the average values (mean ± SEM); the number of measurements is depicted within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; **, p < 9 × 10 −3 ; ***, p < 2 × 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences. The left panels in ( B , C ), designated “-Untag. Type II”, depict the R f and D values of myc-ALK2-WT co-expressed with HA-ALK2-WT without or with IgG αHA crosslinking; these values were taken from A,B and are shown to enable direct comparison with the effects of co-expressing untagged ACVR2A/B. As shown, co-expression with untagged ACVR2A interfered with the formation of homomeric ALK2-WT complexes, which were restored in the presence of ActA (which dimerizes ACVR2A; G). On the other hand, ACVR2B (which forms homodimers also without ActA; E) did not disrupt ALK2-WT homomeric interactions and elevated them in the presence of ActA. Of note, ACVR2B-KD had the same effects as ACVR2B, demonstrating that the kinase activity of the type II receptor is not required.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Labeling, Variant Assay, Comparison, Activity Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is more effective than ACVR2A in mediating ALK2-R206H homomeric interactions. Patch/FRAP studies were conducted exactly as described in , except that myc-and HA-tagged ALK2-R206H replaced the tagged ALK2-WT constructs. The experimental scheme follows the one depicted in , as explained in . The lateral mobility of the Fab’-labeled myc-ALK2-R206H was measured by FRAP. ( A ) The cell surface levels of myc-ALK2-R206H are not altered by co-expression with untagged ACVR2A or ACVR2B. The experiment was conducted as described in A, except that the surface levels of myc-ALK2-R206H were measured by the point confocal method (see A). Data are mean ± SEM of 30 measurements in each case. No significant differences were observed between the surface levels of myc-ALK2-R206H expressed alone or together with untagged ACVR2A or ACVR2B (one-way ANOVA and Bonferroni post hoc test). ( B ) Average R f values; ( C ) average D values. Bars depict the average values (mean ± SEM); the number of measurements is depicted within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (**, p < 8 × 10 −3 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences. The left panels in ( B , C ), designated “-Untag. Type II”, depict the R f and D values of myc-ALK2-R206H co-expressed with HA-ALK2-R206H without or with IgG αHA crosslinking; these values were taken from C,D and are shown to enable direct comparison with the effects of co-expressing untagged ACVR2A/B. Co-expression with untagged ACVR2A had no effect on ALK2-R206H homomeric interactions, which remained undetectable but were induced in the presence of ActA, conditions under which ACVR2A undergoes dimerization ( G). On the other hand, ACVR2B (which forms homodimers also without ActA; E) induced ALK2-R206H homomeric interactions already without ActA. The effects of kinase-dead untagged ACVR2B-KD were indistinguishable from those of ACVR2B.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Construct, Labeling, Expressing, Comparison

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-R206H-mediated pSMAD1/5/8 formation is induced by ACVR2B more efficiently than by ACVR2A. U2OS cells were transfected with vectors encoding HA-ALK2-R206H (or HA-ALK2-WT) alone or with myc-tagged ACVR2A, ACVR2B, or ACVR2B-KD. After 24 h, cells were starved (2 h, 1% serum) and stimulated (or not; control) with ActA (4 nM, 60 min, 37 °C). Cells were lysed, subjected to SDS--PAGE, and immunoblotted for pSMAD1/5/8, tSMAD1/5/8, and β-actin. As shown in , the cell-surface levels of the tagged receptors were similar and were not affected by the co-expressed receptors. ( A , B ) Representative blots of ActA signaling to pSMAD1/5/8. ( C , D ) Quantification of ActA-mediated pSMAD1/5/8 formation. The bands were visualized by ECL and quantified by densitometry. Data are mean ± SEM of the pSMAD1/5/8 over tSMAD1/5/8 ratio of 5 ( C ) or 4 ( D ) independent experiments. The value obtained for ActA-stimulated cells co-transfected with HA-ALK2-R206H and myc-ACVR2B was taken as 1. Asterisks show significant differences between the pairs indicated by brackets, using one-way ANOVA and Bonferroni post hoc test (*, p < 0.02; **, p < 4 × 10 −3 ; ***, p < 8 × 10 −4 ; n.s. = not significant).

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, SDS Page

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is superior to ACVR2A in eliciting ALK2-R206H-mediated transcriptional activation of the SMAD1/5/8 pathway. U2OS cells were co-transfected with BRE-Luc and pRL-TK, together with HA-ALK2-R206H or HA-ALK2-WT (alone or together with myc-ACVR2A, myc-ACVR2B, or myc-ACVR2B-KD). These constructs were replaced by empty vector for control samples. After 17 h, cells were starved without serum (5 h) and stimulated (or not; control) with ActA (2 nM, 19 h). Relative Luminescence Units (RLU) are expressed as mean fold induction ± SEM (n = 4 independent experiments). The results were normalized for transfection efficiency using Renilla luminescence by the DLR luminescence assay. The value in untreated, unstimulated cells was taken as 1. The cell-surface levels of the tagged receptors were not altered by the co-expressed receptors . Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (**, p < 1 × 10 −3 ; ***, p < 5 × 10 −4 ; ****, p < 10 −4 ; n.s. = not significant).

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Luminescence Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: Model for the recruitment of ALK2-R206H into homomeric clusters by ACVR2A/B and its dependence on the dimeric nature of the type II receptors. ( A ) Homodimerization state of the singly-expressed receptors and the dependence on ActA. ACVR2B (red) forms stable homodimers already without ActA, which are enhanced by the ligand (thicker black arrow). ACVR2A (green) requires ActA (orange) to form homodimers. ALK2-WT (light blue) forms homodimers, while ALK2-R206H (dark blue) does not, and both are unaffected by ActA. ( B ) Effect of complex formation with ACVR2A or ACVR2B on the homomeric clustering of ALK2-R206H. The recruitment of the mainly monomeric ALK2-R206H into clusters by the type II receptor depends on the extent of homodimerization of the type II receptor. Thus, the largely dimeric ACVR2B can induce ALK2-R206H clustering already without ligand, while ActA enhances this effect due to increasing ACVR2B homomeric complex formation and its heteromeric interactions with ALK2-R206H. On the other hand, ACVR2A cannot induce clustering of ALK2-R206H, as both receptors are mainly monomeric in the absence of ligand. Upon binding of ActA, ACVR2A forms homodimers and can then induce clustering of ALK2-R206H. The homomeric clustering of ALK2-R206H leads to aberrant signaling to SMAD1/5/8 without a need for phosphorylation by the type II receptor.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic understanding of N -glycosylation in Ebola virus glycoprotein maturation and function

doi: 10.1074/jbc.M116.768168

Figure Lengend Snippet: Comparison of EBOV GP expression and incorporation into E-VLPs. 293T WT, ΔCNX, ΔCRT, and ΔΔ cells were transfected with the indicated FL-GP or ΔMLD-GP expression vectors and an EBOV matrix VP40 expression vector. E-VLPs were recovered from cell culture supernatants and purified by ultracentrifugation. Protein expression in cells and E-VLPs was analyzed by Western blotting.

Article Snippet: A plasmid expressing the full-length EBOV Zaire subtype matrix VP40 was purchased from Sino Biological.

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Purification, Western Blot

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A HIV-1 firefly luciferase reporter viruses pseudotyped with EBOV-GP 1,2 were produced from HEK293T WT, PDIA3, CALR or CANX-overexpressing, and PDIA3 -, CALR - or CANX -KO cells. After infecting HEK293T cells with an equal number of these different viruses, viral entry was determined by measuring intracellular luciferase activities. Viral entry is shown as relative values, with the entry of viruses produced from HEK293T WT cells in the presence of a control vector set to 100%. B EBOV replication and transcription-competent virus-like particles (trVLPs) were produced (p0) and passaged two times (p1, p2) in HEK293T cells in the presence or absence of PDIA3, CALR, or CANX. EBOV replication was determined by measuring intracellular Renilla luciferase activity. Viral replication was also measured in the absence of EBOV-L [L(-)], which served as a negative control. Results from three independent experiments are presented. C Huh7 cells were transfected with increasing amounts of vectors expressing PDIA3, CALR, or CANX, and infected with EBOV Mayinga strain at 0.01 multiplicity of infection (MOI). Viral RNAs were extracted from the supernatants at indicated times and quantified by real-time PCR. D EBOV virus-like particles (VLPs) were produced from HEK293T WT, CALR or CANX-overexpressing, and CALR - or CANX -KO cells after expression of EBOV-GP 1,2 with EBOV-VP40. VLPs were purified by ultra-centrifugation and protein expression in cell lysate and virions was analyzed by WB. GP, VP40, and endogenous CALR and CANX were detected by their specific antibodies; ectopic CALR and CANX were detected by anti-Myc or anti-HA. E Viral fusion proteins from indicated viruses were expressed with CALR in HEK293T cells (lanes 1–14). Alternatively, they were also expressed in HEK293T WT or CALR -KO cells (lanes 15-28). Protein expression was detected by WB. EBOV-GP 1,2 , MERS-S, SRAS1-S, and SARS2-S were detected by anti-FLAG; IAV (H5N5) HA, VSV-G, HIV-1 Env, and CALR were detected by their specific antibodies. F Viral fusion proteins from indicated viruses were expressed with CANX in HEK293T cells (lanes 1-14). Alternatively, they were also expressed in HEK293T WT or CANX -KO cells (lanes 15-28). Protein expression was detected by WB as in D , except that CANX was detected by its specific antibody. Error bars in A , B , and C represent the standard error of measurements (SEMs) calculated from three independent experiments.

Article Snippet: Rabbit polyclonal anti-Zaire EBOV-GP (40442-T48, 1:5000), anti-EBOV-VP40 (40446-T48, 1:10,000), anti-IAV H5HA (11048-RP02, 1:5000), anti-HDAC6 (100768-T08, 1:1000), and anti-RNF26 (203155-T32, 1:500) were purchased from Sino Biological.

Techniques: Luciferase, Produced, Plasmid Preparation, Activity Assay, Negative Control, Transfection, Expressing, Infection, Real-time Polymerase Chain Reaction, Purification, Centrifugation

Journal: bioRxiv

Article Title: High-resolution structure of mammalian PI31–20S proteasome complex reveals mechanism of proteasome inhibition

doi: 10.1101/2023.04.03.535455

Figure Lengend Snippet: HepG2 cells were transfected with pCMV3-FlagPI31 or vehicle controls as described in Experimental Procedures. Extracts from control and PI31 overexpression conditions were prepared and assayed for proteasome activity using peptide substrates for each of the indicated catalytic subunits, as described in Experimental Procedures. Bars are mean (± SD) activities from n=4 lysates of independently transfected cells. Activity in control (-FlagPI31) extracts was set as 100 and other activities within the same experiment are expressed as a percentage of that value. Differences in activity were analyzed by 2-way ANOVA and multiple comparisons within each catalytic subunit were tested by Sidak’s multiple comparisons test. Similar results were obtained in three independent experiments.

Article Snippet: A mammalian expression vector, pCMV3-N-FlagPI31 wt was obtained from Sino Biological Inc (catalog #HG17079-NF).

Techniques: Transfection, Over Expression, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Insight into Molecular Mechanism for Activin A-Induced Bone Morphogenetic Protein Signaling

doi: 10.3390/ijms21186498

Figure Lengend Snippet: Only BMP type I receptor subtype ALK2, but not ALK1/3/6, is able to mediate activin A-induced BMP signaling upon FKBP12 dissociation. ( A ) RT-PCR confirmed that ALK2 is the only predominately expressed BMP type I receptor subtype in HepG2BRA cells and only a trace amount of ALK3 and ALK6 can be detected (note: the y axis is in log scale). The expression level of ALK1 was set as 1, and all the other numbers were normalized with ALK1. ( B ) The specific siRNA effectively knocked down ALK2 in HepG2BRA cells. ( C ) None of the three BMP type I receptor subtypes (ALK1/3/6) could rescue the luciferase downregulation caused by ALK2 knockdown in the presence of activin A and FK506, suggesting that ALK1/3/6 cannot signal activinA-induced BMP signaling upon FKBP12 dissociation, and ( D ) this result was confirmed by Western blotting. Results of luciferase assay and the RT-PCR assay were represented as a mean relative luciferase units (RLU) ± SEM and relative quantity (RQ) ± SEM ( n = 3, * p < 0.05, ** p < 0.01).

Article Snippet: The following plasmids were used in the current study: pCDNA3 plasmids harboring ALK2 WT , ALK3 WT and ALK6 WT were purchased from Addgene (#80870, #80873 and #80882). pCMV3 plasmids harboring activin A receptor type 2A (ActR2A) and human activin A receptor type 2B (ActR2B) were obtained from Sino Biological Inc.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Luciferase, Knockdown, Western Blot

Journal: Biology Methods & Protocols

Article Title: A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

doi: 10.1093/biomethods/bpad026

Figure Lengend Snippet: Flow diagram of the purification of C-terminal FLAG-tagged ASNS protein. Beginning with HEK 293T cells stably expressing WT or ASNS protein variants, the schematic shows the steps utilized to immunoprecipitate and purify C-terminal FLAG-tagged ASNS protein using gel-immobilized M2 anti-FLAG antibody. Protein purity was assessed by SDS–PAGE separation and staining with Coomassie Blue.

Article Snippet: A WT ASNS expression plasmid was obtained from Sino Biological (HG16454-CF) and contains a C-terminal linker and FLAG-tag sequence following the ASNS open reading frame.

Techniques: Purification, Stable Transfection, Expressing, SDS Page, Staining

Journal: Biology Methods & Protocols

Article Title: A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

doi: 10.1093/biomethods/bpad026

Figure Lengend Snippet: Stability of purified WT C-terminal FLAG-tagged ASNS. ASNS activity of purified WT ASNS-FLAG was measured after storage at either −80°C or 4°C for 10 days ( N = 3 per group). Day 1 was defined as the day following purification and quantification.

Article Snippet: A WT ASNS expression plasmid was obtained from Sino Biological (HG16454-CF) and contains a C-terminal linker and FLAG-tag sequence following the ASNS open reading frame.

Techniques: Purification, Activity Assay

Journal: Biology Methods & Protocols

Article Title: A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

doi: 10.1093/biomethods/bpad026

Figure Lengend Snippet: Modeling and over-expression of ASNS-FLAG variants associated with ASNSD. ( A ) Using the reported human ASNS crystal structure, the two domains, shown are the N-terminal domain that has glutamine binding and glutaminase activity and the C-terminal domain that catalyzes the synthesis of asparagine from aspartate and ATP. The locations of the R49Q, G289A, and T337I variants are shown in red. The C-terminal bound AMP product and the N-terminal bound glutamine substrate are shown with backbones in green, nitrogen atoms in blue, and oxygen atoms in red to illustrate the relative proximity of each variant to the substrate binding pockets. ( B ) Immunoblot analysis with FLAG antibody showing the relative levels of WT, R49Q, G289A, and T337I ASNS-FLAG variants for individual stably expressing HEK 293T cell lines.

Article Snippet: A WT ASNS expression plasmid was obtained from Sino Biological (HG16454-CF) and contains a C-terminal linker and FLAG-tag sequence following the ASNS open reading frame.

Techniques: Over Expression, Binding Assay, Activity Assay, Variant Assay, Western Blot, Stable Transfection, Expressing

Journal: Biology Methods & Protocols

Article Title: A method for measurement of human asparagine synthetase (ASNS) activity and application to ASNS protein variants associated with ASNS deficiency

doi: 10.1093/biomethods/bpad026

Figure Lengend Snippet: Purification and activity analysis of ASNS WT and variants. ( A ) Summary of the starting protein content, concentrations after purification, and total yield of ASNS proteins prepared from HEK 293T cells stably expressing WT, R49Q, G289A, or T337I ASNS-FLAG. ( B ) SDS – PAGE of each purified ASNS-FLAG protein. Purified bands are seen at the expected 64 kDa for ASNS-FLAG protein. ( C ) Comparison of WT, R49Q, G289A, or T337I ASNS enzymatic activity. All assays were performed in triplicate within an experiment to evaluate variation and each experiment was repeated with an independent protein preparation to ensure reproducibility. Data are presented as means ± standard deviations and an asterisk denotes a P ≤.01 compared to the WT ASNS value.

Article Snippet: A WT ASNS expression plasmid was obtained from Sino Biological (HG16454-CF) and contains a C-terminal linker and FLAG-tag sequence following the ASNS open reading frame.

Techniques: Purification, Activity Assay, Stable Transfection, Expressing, SDS Page, Comparison

Journal: Science Advances

Article Title: A phospho-tyrosine–based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity

doi: 10.1126/sciadv.abq0084

Figure Lengend Snippet: ( A and B ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. The SBM in CSK and its mutation in CSK-AAA are indicated. ( C ) FA competition binding assay. Peptides containing the candidate SBM of CSK and the mutated sequence CSK-AAA compete with fluorescein-Puc 91–106 for binding to the SPOP MATH domain. [L] is the peptide concentration. Continuous lines are nonlinear least-squares fits to a complete competitive binding model. See for K d values. ( D ) Clustal Omega–based sequence alignment of Src family kinases and CSK. The SBM and the conserved tyrosine are highlighted by green and red boxes, respectively. ( E ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. ( F ) Nuclear translocation assay of SPOP-deficient RAW264.7 cells that were reconstituted with indicated forms of SPOP. ( G ) qPCR analysis of cells described in (F) that were stimulated with CpG-DNA for 4 hours. ( H to K ) In vitro kinase assays based on proteins expressed in HEK293T cells. CSK-AAA, SST340/341/341AAA. Fold differences of CSK autophosphorylation as determined by phosphor image analysis. The activity used for reference (set as 1) is underlined. Data represent mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.005 are determined by two-way ANOVA with Sidak’s multiple comparison test (G).

Article Snippet: N-terminal FLAG-tagged mouse CSK-WT and CSK-SST340/341/342AAA were expressed using the vector pCMV3-N-FLAG (Sino Biological). pcDNA3-N-FLAG-hIRF8 was from Sino Biological.

Techniques: Mutagenesis, Binding Assay, Sequencing, Concentration Assay, Nuclear Translocation Assay, In Vitro, Activity Assay