Journal: Proteomes

Article Title: Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

doi: 10.3390/proteomes6040042

Figure Lengend Snippet: In vitro phosphorylation of α4/β2 nAChRs by CaMKII or PKA. The α4 and β2 nAChR subunits were co-expressed in HEK cells, isolated by immunoprecipitation, and subjected to mass spectrometry. Phosphorylation level was normalized to total subunit protein. ( a ) At baseline, there was a high level of phosphorylation of S470, S530, and S540 on the α4 subunit, and incubation with lambda phosphatase dephosphorylated S540 and S543 to undetectable levels. ( b ) Incubation with CaMKIIα in the presence of calcium and calmodulin increased phosphorylation of T417 and S468 on the α4 subunit significantly. ( c ) Incubation with PKA in the presence of cyclic AMP increased phosphorylation of S470, S491, and S521 significantly. * p < 0.05; *** p < 0.005. Error bars represent standard error of the mean; n = 6/condition.

Article Snippet: The remaining three groups were harvested in the absence of phosphatase inhibitors, and immunoprecipitated receptors were subject to in vitro dephosphorylation with purified lambda phosphatase (New England Biolabs, Ipswich, MA, USA; all but baseline group received this treatment).

Techniques: In Vitro, Isolation, Immunoprecipitation, Mass Spectrometry, Incubation

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. Western blot analysis of the expression levels of C16orf74 in pancreatic cancer cell lines. Control: Flag-tagged C16orf74-overexpressed diluted cell lysate. B. Phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by Western blot analysis using an anti-C16orf74 polyclonal antibody. The upper band disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). C. Phosphorylation at threonine 44 (T44) of C16orf74. Flag-tagged wild type (WT), T41A and T44A mutants of C16orf74 were used to transfect COS-7 cells. The phosphorylated form of wild-type C16orf74 (arrow) was disappeared in the T44A mutant. D. Immunocytochemical analysis in a pancreatic cancer cell line (PK-1) using the anti-C16orf74 antibody, demonstrating the plasma membrane localization of endogenous C16orf74 (Green). DAPI staining is shown in blue.

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: Western Blot, Expressing, Control, Incubation, Phospho-proteomics, Mutagenesis, Clinical Proteomics, Membrane, Staining

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. In vitro exogenous association of C16orf74 and PPP3CA. The Flag-tagged C16orf74 construct or vector alone was cotransfected with a myc-tagged PPP3CA construct into HEK293 cells. Cell lysates were immunoprecipitated using mouse anti-Flag antibody (left) or anti-myc antibody (right). Immunoblotting of the immunoprecipitates with rabbit anti-Flag or anti-myc antibodies revealed a specific interaction between the phosphorylated form of C16orf74 (arrow) and PPP3CA. B. In vitro endogenous association of C16orf74 and PPP3CA from Capan-1 pancreatic cancer cells, which endogenously express high levels of both C16orf74 and PPP3CA. Capan-1 cell lysates were immunoprecipitated using anti-C16orf74 antibody (left) or anti- PPP3CA antibody (right). Immunoblotting of the immunoprecipitates with anti-C16orf74 antibody or anti-PPP3CA antibodies revealed a specific interaction between C16orf74 and PPP3CA. Endogenous PPP3CA interacted with the phosphorylated form of endogenous C16orf74 (arrow). C. Interactions of wild-type C16orf74 (WT) and mutants of C16orf74 with PPP3CA, as assessed by IP analysis. Expression vectors for myc-His-tagged PPP3CA and Flag-tagged C16orf74 constructs were doubly transfected into HEK293T cells. C16orf74 (anti-Flag) was IP, and the indicated molecules were immunoblotted (IB) in western blot analysis. WT, replacement (T44A; non-phosphorylated form of C16orf74) and deletion mutants (∆PDIIIT; deletion mutant of PPP3CA binding motif) were analyzed. PPP3CA bound to wild-type C16orf74 but not the non-phosphorylated form of C16orf74 or the deletion mutant of the PPP3CA binding motif. D. Subcellular localization of C16orf74 (wild type or ∆PDIIIT) and PPP3CA in mammalian cells. Flag-tagged (green) C16orf74 (wild type or ∆PDIIIT) and myc-tagged (red) PPP3CA constructs were cotransfected into COS-7 cells and subjected to immunocytochemical staining. Flag-C16orf74 (wild type) and myc-PPP3CA colocalized on the under the cytoplasmic membrane of COS-7 cells (yellow), but Flag-C16orf74 (∆PDIIIT) did not colocalize with myc-PPP3CA, which was present diffusely in the cytoplasm. E. Interactions of endogenous C16orf74 with PPP3CA as assessed by IP analysis. The phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by western blot analysis using an anti-C16orf74 polyclonal antibody. Pre IP (left; non-immunoprecipitated by PPP3CA), the phosphorylated form of C16orf74 (upper band) disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). Immunoprecipitation by PPP3CA (right) revealed that the phosphorylated form of C16orf74 (upper band) interacted with PPP3CA, whereas the non- phosphorylated form of C16orf74 did not. F. Invasion activity of wild-type C16orf74 (WT) and the two mutants (T44A: non-phosphorylated form of C16orf74; and ∆PDIIIT, deletion mutant of the PPP3CA binding motif). The WT-C16orf74 expression vector, T44A-C16orf74 expression vector, ∆PDIIIT-C16orf74 expression vector, and Mock vector were each transfected into NIH3T3 cells. The Matrigel invasion assay revealed an enhanced cell number for WT-C16orf74-over-expressing cells (3.4-fold, * P = 0.013) but not so enhanced for ∆PDIIIT-C16orf74-over-expressing cells (1.4-fold, ** P = 0.017) or T44A-C16orf74-over-expressing cells (2.3-fold,*** P = 0.038).

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: In Vitro, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Staining, Membrane, Incubation, Activity Assay, Invasion Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagram of domains and motifs of human doublecortin-like kinase 1 (DCLK1) (UniProt O15075) that are conserved in the mouse DCLK1 used in this study. DC1, N-terminal doublecortin-like (DCX) domain; DC2, C-terminal DCX domain; kinase domain. Motifs enriched in PEST (proline/P, glutamic acid/E, serine/S, threonine/T) and DEND (aspartic acid/D, glutamic acid/E, asparagine/N, aspartic acid/D) based on and . Below: model of human DCLK1. DC1 domain (1mg4; ), DC2 domain modeled by homology to DCX-DC2 (5ip4; ), kinase domain (5jzj; ). DCLK1 is shown as a full-length pseudo-model, with projection domains/tails (and domain linkers) modeled as unfolded to visualize the length and convey the intrinsic disorder predicted for those regions. The mouse DCLK1 (1–740) used in this paper has the same amino acid boundaries as that of humans. ( B ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified wild-type (WT), ΔC, and kinase-dead (D511N) DCLK1 proteins separated by phosphorylation level. Representative gel from n = 3 independent experiments. ( C ) Total internal reflection fluorescence microscopy (TIRF-M) images of 3 nM and 25 nM sfGFP-DCLK1 WT, ΔC, and D511N (green), expressed in bacteria under standard conditions, binding to taxol-stabilized microtubules (blue). Scale bars: 2.5 μm. ( D ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 2748.9 ± 2073.6 for 3 nM WT, 16119.6 ± 4324.3 for 25 nM WT, 1.2 ± 31.6 for 3 nM ΔC, 3.8 ± 101.9 for 25 nM ΔC, 9072.4 ± 3380.1 for 3 nM D511N, and 19666.6 ± 3345.3 for 25 nM D511N (n>100 microtubules from n = 3 independent trials for each concentration of each protein). Gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial. ***p<0.0001 and p = 0.3240 for 25 nM WT vs 25 nM D511N, calculated using Student’s t-test. p-values were calculated using n = 3. ( E ) Coomassie blue-stained SDS-PAGE Phos-tag gel of purified DCLK1-WT and -ΔC incubated with lambda phosphatase (λPP) or incubated in buffer alone for 1 hr at 30°C. Representative gel from n = 3 independent experiments. ( F ) TIRF-M images of 3 nM sfGFP-DCLK1 WT and ΔC (green) after treatment with λPP, binding to taxol-stabilized microtubules (blue). Scale bars: 2.5 μm. ( G ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 15090.7 ± 5285.6 for 3 nM WT + λPP, 18155.3 ± 3833.5 for 25 nM WT + λPP, 12004.2 ± 3490.3 for 3 nM ΔC + λPP, and 21240.6 ± 3413.5 for 25 nM ΔC + λPP (n>100 microtubules from n = 3 independent trials for each protein concentration; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial.). D511N data are reproduced from ( D ) for comparison. p = 0.3566 for 25 nM WT + λPP vs 25 nM ΔC + λPP, p = 0.6341 for 25 nM WT + λPP vs 25 nM D511N, p = 0.5989 for 25 nM ΔC + λPP vs 25 nM D511N, and p = 0.4462 for 3 nM WT + λPP vs 3 nM ΔC + λPP, calculated using Student’s t-test. p-values were calculated using n = 3. For all experiments, at least two separate protein purifications were used. Figure 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Fluorescence, Microscopy, Bacteria, Binding Assay, Concentration Assay, Incubation, Protein Concentration, Comparison

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of sfGFP-DCLK1 proteins used in this study. ( B ) Anion exchange elution chromatograms for wild-type doublecortin-like kinase 1 (DCLK1-WT) compared to -T687A, -T688A, and -D511N (left) and DCLK1-ΔC compared to ∆C DC1-4A and ∆C DC2-4A (right), showing that all proteins elute in a homogenous peak at approximately the same ionic strength, revealing that they are similarly folded. ( C ) Anion exchange elution chromatograms for DCLK1-WT and DCLK1-ΔC prepped in the presence or absence of lambda phosphatase (λPP). The non-phosphorylated proteins (+λPP) elute at a lower ionic strength consistent with a lower net negative charge. Note the larger shift in the elution volume for the non-phosphorylated vs phosphorylated DCLK1-ΔC, indicating a more pronounced change in the net charge between the two preps. Figure 1—figure supplement 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 1—figure supplement 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Immunoblots of strepII-sfGFP-tagged wild-type (WT) or kinase-dead (D511N) doublecortin-like kinase 1 (DCLK1) incubated in the absence or presence of ATPγS for 30 min at 37°C. DCLK1-WT, but not DCLK1-D511N, robustly autophosphorylates. ( B ) Immunoblots of strepII-sfGFP-tagged DCLK1-D511N incubated with an untagged version of DCLK1-WT to distinguish the proteins by size in the absence or presence of ATPγS for 30 min at 37°C. While DCLK1-WT autophosphorylated, there was still no detectable level of phosphorylation for DCLK1-D511N. For ( A ) and ( B ), primary antibodies used were mouse anti-strep (Fisher NBP243719) and rabbit anti-thiophosphate ester (Abcam ab133473). For both ( A ) and ( B ), these blots are representative images from at least n = 3 independent experiments. ( C ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified DCLK1-WT, -ΔC, and -D511N incubated with lambda phosphatase (λPP) or incubated in buffer alone for 1 hr at 30°C. Representative gel from n = 3 independent experiments. ( D ) Representative total internal reflection fluorescence microscopy (TIRF-M) image of 10 nM sfGFP-DCLK1-ΔC (green, co-expressed in bacteria with λPP) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the presence of 2 mM adenosine triphosphate (ATP). Scale bar: 5 μm. ( E ) Coomassie blue-stained SDS-PAGE shows the binding behavior of 500 nM DCLK1-WT or -ΔC in the absence or presence of 2 mM ATP in the absence or presence of 2 μM taxol-stabilized microtubules. In the absence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 99.3 ± 0.5% for WT and 96.3 ± 1.4% for ΔC (n = 3 independent experiments; p = 0.0250). In the presence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 86.2 ± 4.9% for WT and 6.9 ± 5.0% for ΔC (n = 3 independent experiments; p<0.0001). For all experiments, at least two separate protein purifications were used. Figure 1—figure supplement 2—source data 1. Uncropped blots ( A, B ), gels ( C, E ) for the associated panels in . The red box indicates how the blot of gel was cropped. Figure 1—figure supplement 2—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Western Blot, Incubation, Phospho-proteomics, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Fluorescence, Microscopy, Bacteria, Binding Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified doublecortin-like kinase 1 wild-type (DCLK1-WT) and -ΔC proteins separated by phosphorylation level. The first and fifth lanes contain DCLK1-WT and -ΔC expressed in bacteria under standard conditions. All other lanes contain DCLK1-WT and -ΔC that were co-expressed with lambda phosphatase (λPP), which was subsequently separated from DCLK1. Incubation of λPP-treated DCLK1-WT and -ΔC with 2 mM adenosine triphosphate (ATP) at the indicated times reveals a band shift, indicative of an increase in phosphorylation. ( B ) Quantification of the average percent of total DCLK1 protein that is phosphorylated in each condition. Averages are derived from n = 3 independent experiments. ( C ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1-WT and -ΔC (co-expressed in bacteria with λPP) at indicated concentrations (green) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( D ) Quantification of microtubule-bound sfGFP-DCLK1-WT fluorescence intensity plotted against concentration after a 30-min incubation in the absence or presence of ATP (WT without ATP, K D = 2.1 nM, and WT with ATP, K D = 5.4 nM, derived from at least n = 3 independent trials per condition). ( E ) Quantification of microtubule-bound sfGFP-DCLK1-ΔC fluorescence intensity plotted against concentration after a 30-min incubation in the absence or presence of ATP (ΔC without ATP, K D = 3.9 nM, and ΔC with ATP, K D = 161.0 nM, derived from n = 3 independent trials). ( F ) TIRF-M images of 10 nM sfGFP-DCLK1-WT or -ΔC (green, co-expressed in bacteria with λPP) binding to non-stabilized GDP microtubules grown from GMPCPP seeds (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( G ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 15004.4 ± 6503.8 for WT, 12535.9 ± 3247.5 for WT + ATP, 12111.3 ± 3534.0 for ΔC, and 1579.3 ± 866.5 for ΔC + ATP (n>60 microtubules from n = 2 independent trials for each condition; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.6360 for WT vs ΔC and p = 0.0440 for WT + ATP vs ΔC + ATP, calculated using Student’s t-test; p-values were calculated using n = 2). For all experiments, at least two separate protein purifications were used. Figure 2—source data 1. Uncropped gel for the associated panel in . The red box indicates how the gel was cropped. Figure 2—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Bacteria, Incubation, Electrophoretic Mobility Shift Assay, Derivative Assay, Fluorescence, Microscopy, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagrams depicting the domains, amino acid boundaries, and mutations relevant to the doublecortin-like kinase 1 (DCLK1) constructs used. ∆C DC1-4A indicates the four residues in DC1 that were mutated to alanines S77, S83, S96, and T143. ∆C DC2-4A indicates the four residues in DC2 that were mutated to alanines T189, S193, T218, and S228. ( B ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1 ΔC, ∆C DC1-4A , and ∆C DC2-4A , co-expressed in bacteria with lambda phosphatase (λPP), at indicated concentrations binding to taxol-stabilized microtubules (blue) in the absence or presence of adenosine triphosphate (ATP). Scale bars: 2.5 μm. ( C ) Quantification of microtubule-bound 5 nM sfGFP-DCLK1 fluorescence intensity. For 5 nM concentrations in the absence of ATP, means ± sd: 12883.5 ± 2881.6 for ΔC, 12245.5 ± 3283.0 for ∆C DC1-4A , 8552.7 ± 2097.3 for ∆C DC2-4A (n>100 microtubules per condition from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.8128 for ΔC vs ∆C DC1-4A and p = 0.1031 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). For 5 nM concentrations in the presence of ATP, means ± sd: 987.6 ± 202.5 for ΔC + ATP, 4042.6 ± 1624.6 for ∆C DC1-4A + ATP, 2482.0 ± 1058.3 for ∆C DC2-4A + ATP (n>100 microtubules from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.0319 for ΔC vs ∆C DC1-4A and p = 0.0742 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). ( D ) Quantification of microtubule-bound 20 nM sfGFP-DCLK1 fluorescence intensity. For 20 nM concentrations in the absence of ATP, means ± sd: 23634.3 ± 1725.1 for ΔC, 22277.3 ± 1334.9 for ∆C DC1-4A , 19912.2 ± 5408.6 for ∆C DC2-4A (n>100 microtubules per condition from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.3419 for ΔC vs ∆C DC1-4A and p = 0.3196 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). For 20 nM concentrations in the presence of ATP, means ± sd: 1579.8 ± 585.1 for ΔC + ATP, 12556.9 ± 3419.9 for ∆C DC1-4A + ATP, 3503.4 ± 826.7 for ∆C DC2-4A + ATP (n>100 microtubules from n = 4, 6, and 5 independent trials for ΔC, ∆C DC1-4A , and ∆C DC2-4A , respectively; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.0002 for ΔC vs ∆C DC1-4A and p = 0.0058 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = number of independent trials as stated above). For all experiments, at least two separate protein purifications were used.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Construct, Fluorescence, Microscopy, Bacteria, Binding Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagram depicting the domains, amino acid boundaries, and mutations in the C-terminal region relevant to the doublecortin-like kinase 1 (DCLK1) constructs used. ( B ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified kinase-dead (D511N), WT, T687A, and T688A DCLK1 proteins separated by phosphorylation level. For all experiments, DCLK1 proteins were co-expressed with lambda phosphatase (λPP), which was subsequently separated from DCLK1. Incubation of λPP-treated DCLK1 proteins with 2 mM adenosine triphosphate (ATP) at the indicated times reveals a shift in the phosphorylation level to varying degrees. ( C ) Quantification of the average percent of total DCLK1 protein that is phosphorylated in each condition. Averages were derived from n = 3 independent experiments. ( D ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1-WT, -T687A, and -T688A (co-expressed in bacteria with λPP) at indicated concentrations (green) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( E ) Quantification of microtubule-bound sfGFP-DCLK1-WT, -T687A, and -T688A fluorescence intensity plotted against concentration after a 30-min incubation in the absence of ATP (K D = 3.0 nM, 2.8 nM, and 2.6 nM for WT, T687A, and T688A, respectively, from at least n = 3 independent trials per condition). ( F ) Quantification of microtubule-bound sfGFP-DCLK1-WT, -T687A, and -T688A fluorescence intensity plotted against concentration after a 30-min incubation with ATP (K D = 5.7 nM, 3.9 nM, and 239.2 nM for WT, T687A, and T688A, respectively, from at least n = 3 independent trials per condition). ( G ) Coomassie blue-stained SDS-PAGE shows the binding behavior of 500 nM DCLK1-WT or -T688A in the absence or presence of 2 mM ATP in the absence or presence of 2 μM taxol-stabilized microtubules. In the absence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 99.3 ± 0.5% for WT and 98.9 ± 1.0% for T688A (n = 3 independent experiments; p = 0.5690). In the presence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 86.2 ± 4.9% for WT and 7.9 ± 3.5% for T688A (n = 3 independent experiments; p<0.0001). For all experiments, at least two separate protein purifications were used. Figure 5—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 5—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Construct, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Incubation, Derivative Assay, Fluorescence, Microscopy, Bacteria, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels of purified doublecortin-like kinase 1 (DCLK1) proteins fractionated by sucrose density-gradient centrifugation. Representative gels from n = 2 independent experiments for each protein are shown. ( B ) Quantification of the average percent of DCLK1 protein in each fraction, revealing a peak in fraction 4 for all DCLK1 proteins. ( C ) Coomassie blue-stained SDS-PAGE gel of purified DCLK1-WT and -T688A proteins co-expressed with lambda phosphatase (λPP) in bacteria, which was first incubated either in the absence or in the presence of adenosine triphosphate (ATP) for 30 min at 25°C followed by an incubation with calpain for 15 min at 30°C. Similar band patterns are seen for each protein under each condition after cleavage with calpain. Gel is representative of n = 3 independent experiments. Figure 5—figure supplement 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 5—figure supplement 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Gradient Centrifugation, Bacteria, Incubation

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet:

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Microscopy, Recombinant, Software

Journal: Developmental cell

Article Title: Phosphorylation of Ci/Gli by Fused family kinases promotes Hedgehog signaling

doi: 10.1016/j.devcel.2019.06.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Anti-Flag antibody Millipore Sigma Clone ID: M2; Cat#1804; RRID: AB_439685 Mouse Anti-HA (F-7) antibody Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 Mouse Anti-Myc antibody Santa Cruz Biotechnology Clone ID: 9E10; Cat#sc-40; RRID: AB_627268 Rabbit Anti-Myc antibody Abcam Cat#ab9106; RRID: AB_307014 Rat Anti-Ci antibody DSHB Cat#2A1; RRID: AB_2109711 Mouse Anti-Fu antibody DSHB Cat#22F10; RRID: AB_528258 Goat Anti-PKC ζ antibody Santa Cruz Biotechnology Clone ID: c-20; Cat#sc-216; RRID: AB_2300359 Mouse Anti-α-tubulin antibody Millipore Sigma Clone ID: DM1A; Cat#T9026; RRID: AB_477593 Mouse Anti-GFP antibody Takara Biol Cat#632380; RRID: AB_10013427 Mouse Anti-acetylated Tubulin antibody Millipore Sigma Clone ID:6-11b1; Cat#T7451; RRID: AB_609894 Goat Anti-Gli2 antibody R&D Systems Cat#AF3635; RRID: AB_2111902 Goat Anti-Gli3 antibody R&D Systems Cat#AF3690; RRID: AB_2232499 Bacterial and Virus Strains NEB5-Alpha Competent E. Coli New England Biolabs Cat# {"type":"entrez-nucleotide","attrs":{"text":"C29871","term_id":"2361667","term_text":"C29871"}} C29871 BL21 Competent E.Coli New England Biolabs Cat#C2530H Chemicals, Peptides, and Recombinant Proteins Leptomycin B Millipore Sigma Cat#L2913 Acti-Stain 670 phalloidin Cytoskeleton Cat#PHDN1-A Bluo-Gal Thermo Fisher Scientific Cat#15519028 L-Glutathione Reduced Millipore Sigma Cat#G4251 SAG Millipore Sigma Cat#566660 DAPI Millipore Sigma Cat#D9542 ATP [γ − 32 P] Perkin Elmer Cat#BLU002A Insulin Millipore Sigma Cat#I6634 Fetal Bovine Serum Millipore Sigma Cat#F4135 Fetal Bovine Serum Millipore Sigma Cat#F0926 Calf Bovine Serum Iron Fortified ATCC Cat#ATCC30-2030 3× Flag Peptide Millipore Sigma Cat#F4799 HA Synthetic Peptide Thermo Fisher Scientific Cat#26184 Casein Kinase I New England Biolabs Cat#P6030 Recombinant Human Sonic Hedgehog Protein (Shh-N) R&D Systems Cat#8908-HSH/CF Lambda Protein Phosphatase New England Biolabs Cat#P0753 Critical Commercial Assays Dual Luciferase Reporter Assay System Promega Cat#E1910 MEGAscript T7 High Yield Transcription Kit Thermo Fisher Scientific Cat#AM1334 pIMAGO-biotin Phosphoprotein Detection Kit Millipore Sigma Cat#67221 Anti-Flag M2 Affinity Gel Millipore Sigma Cat#A2220 Pierce Anti-HA Argarose Thermo Fisher Scientific Cat#26181 Glutathione Sepharose 4B GE Healthcare Cat#17075601 SimplyBlue SafeStain Thermo Fisher Scientific Cat#LC6060 RNeasy Plus Mini Kit Qiagen Cat#74134 High Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Fast SYBR Green Master Mix Applied Biosystems Cat#4385612 CellTiter-Glo Luminescent Cell Viability Assay Promega Cat#G7572 Experimental Models: Cell Lines Mouse: NIH/3T3 ATCC Cat#ATCC CRL-1658 Human: HEK293T/17 ATCC Cat#ATCC CRL-11268 D. melanogaster .

Techniques: Recombinant, Luciferase, Reporter Assay, SYBR Green Assay, Cell Viability Assay, Knock-In, Software

Journal: Nucleic Acids Research

Article Title: CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling

doi: 10.1093/nar/gkad298

Figure Lengend Snippet: CSNK2B interacts with IRF1 and regulates its antiviral activity independently of phosphorylation. ( A ) 293FT cell lysates expressing IRF1-FLAG or empty vector was immunoprecipitated with anti-FLAG M2 antibody. Proteins eluted from the precipitates were subjected to an SDS-PAGE followed by western blotting with specific antibodies against CK2 components. ( B ) Pull-down analysis showing direct interaction between purified IRF1-FLAG and recombinant human CSNK2B (rCSNK2B) proteins. ( C ) Phos-tag SDS-PAGE of FLAG-tagged IRF1. PH5CH8 cell lysates were subjected to immunoblotting before (−) and after (+) digestion with lambda protein phosphatase (λ PPase). An arrowhead shows phospho-IRF1. ( D , E ) Phos-tag gel analysis of ectopically expressed IRF1-FLAG in 293FT cells ( D ) or endogenously expressed IRF1 in PH5CH8 cells ( E ). Means ± S.D. of values for abundance of phospho-IRF1 relative to non-phosphorylated IRF1 are shown on right ( n = 3). ( F ) PH5CH8 cells were transfected with siRNAs targeting CK components and infected 48 h later with HAV at an m.o.i. of 10. Percentage of HAV RNA levels relative to non-target control siRNA was determined 4 d.p.i. by RT-qPCR. Immunoblots showing depletion of catalytic CK2 subunits are shown on right. ** P < 0.01 versus control ( n = 3 or 4, one-way ANOVA with Dunnett's multiple comparisons test). ( G ) Immunoblots showing CSNK2B protein abundance in Huh-7.5 cells treated with 10 μM CX-4945 for 24 h. ( H ) Effects of CX-4945 on replication of HAV/NLuc (18f/NLuc) in Huh-7.5 cells and cell viability. ** P < 0.01, *** P < 0.0001 versus control ( n = 3, one-way ANOVA with Bonferroni's multiple comparisons test). ( I ) Effects of CX-4945 on HAV replication and PLAAT4 expression in IRF1 -depleted and control Huh-7.5 cells. ** P < 0.01, *** P < 0.0001 ( n = 3, two-way ANOVA with Sidak's multiple comparisons test). ( J ) NLuc reporter analysis of Huh-7.5 cells expressing NLuc reporter (pNL-4×IRF1). Cells were transfected with indicated siRNAs for 48 h, and relative NLuc values secreted at 48–72 h post-transfection are shown. Immunoblots showing depletion of each siRNA target are shown on right. * P < 0.05, ** P < 0.01 versus control ( n = 4, one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Lambda protein phosphatase was obtained from Bio Academia. miR-122 mimics were synthesized by Dharmacon and transfected by electroporation as miRNA/miRNA* duplexes as described ( ).

Techniques: Activity Assay, Phospho-proteomics, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Purification, Recombinant, Transfection, Infection, Control, Quantitative RT-PCR, Quantitative Proteomics

Journal: Nucleic Acids Research

Article Title: CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling

doi: 10.1093/nar/gkad298

Figure Lengend Snippet: CSNK2B regulates transcription and phosphorylation of AFAP1 that lowers permissiveness to flavivirus replication. ( A ) Immunoblots of AFAP1 and GAPDH as loading control in lysates of PH5CH8 cells transfected with indicated siRNAs, with and without IFNγ stimulation. ( B ) Immunoblots of AFAP1, IRF1 and GAPDH in lysates of PH5CH8 cells depleted of either (or both) CSNK2B and IRF1. Quantitation of AFAP1 protein abundance is shown on the right. ** P < 0.01, *** P < 0.0001 ( n = 3, one-way ANOVA with Dunnett's multiple comparisons test). ( C ) Immunoblots of AFAP1 and GAPDH in lysates of PH5CH8 cells treated with either lambda protein phosphatase (λ PPase, left panels) or indicated concentrations of CX-4945 (right panels). ( D ) Immunoblots of AFAP1 and Src in lysates of PH5CH8 cells treated with 10 μM CX-4945. ( E ) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs targeting AFAP1 (left panels) or CSNK2B (right panels). ( F ) Scheme of CSNK2B-regulated AFAP1 signaling cascades that lead to Src activation. ( G ) Immunoblots of PH5CH8 cell lysates transfected with indicated siRNAs (top). DENV RNA levels were determined at 48 h p.i. (bottom). * P < 0.05 ( n = 3, two-tailed Student's t -test). ( H ) Immunoblots of AFAP1 in lysates of PH5CH8 versus Huh-7.5 cells stably transduced with AFAP1 (top). Huh-7.5 cells stably expressing AFAP1 or vector control were challenged with DENV/NLuc (bottom). NLuc activities at the indicated time points post-infection are shown. ** P < 0.01 ( n = 3, two-way ANOVA with Sidak's multiple comparisons test). ( I ) PH5CH8 cells were transfected with indicated siRNAs and infected 48 h later with dengue virus (DENV) at an m.o.i. of 0.1. CX-4945 (5 μM) was added 2 h post-infection. Infectious titers were determined 48 h p.i. by focus formation assays. FFU, focus forming units. ** P < 0.01, *** P < 0.0001 versus control ( n = 3, one-way ANOVA with Dunnett's multiple comparisons test). ( J ) PH5CH8 cells were infected with DENV or Zika virus (ZIKV) as in (I) and viral RNA levels determined by RT-qPCR. ** P < 0.01 ( n = 3, two-tailed Student's t -test).

Article Snippet: Lambda protein phosphatase was obtained from Bio Academia. miR-122 mimics were synthesized by Dharmacon and transfected by electroporation as miRNA/miRNA* duplexes as described ( ).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Quantitation Assay, Quantitative Proteomics, Activation Assay, Two Tailed Test, Stable Transfection, Transduction, Expressing, Plasmid Preparation, Infection, Virus, Quantitative RT-PCR