active human pkcα  (Addgene inc)

Journal: Nature Communications

Article Title: Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms

doi: 10.1038/s41467-022-29381-7

Figure Lengend Snippet: a – d Immunoblots for the indicated proteins in total cell lysates (input) or immunoprecipitates (IP) with anti-ULK1 or RIgG (negative control) antibodies from ( a ) HEL, ( b ) SET-2, ( c ) KT-1, and ( d ) U937 cells, left untreated or treated with IFNα or IFNβ (10 4 IU/mL) for 10 min, as indicated. Blots are representative of two independent experiments for each cell line. RIgG, normal Rabbit Immunoglobulin G. IP samples and corresponding total cell lysates (input) were resolved and blotted from the same gel as shown in Supplementary Information (uncropped blots). e KT-1 cells were starved overnight and then treated with IFNβ (10 4 IU/mL) for 10 or 30 min, as indicated. After cell lysis, equal amounts of protein were immunoprecipitated (IP) with either PKCδ antibody or control normal rabbit immunoglobulin G (RIgG), as indicated. In vitro kinase assays to assess PKCδ activity were subsequently performed on the immunoprecipitates, using GST-ULK1 recombinant inactive protein as an exogenous substrate. ( Top panel ) Autoradiography film demonstrating PKCδ-induced phosphorylation of ULK1 after IFNβ treatment. ( Bottom panels ) Immunoblots demonstrating total GST-ULK1 protein and total immunoprecipitated PKCδ expression used in each condition for the in vitro kinase assay. Note: a lane between ULK1 and RIgG immunoprecipitates was loaded with 1x loading dye for best separation between the wells. Kinase assay shown is representative of three independent experiments. *unspecific band. f cDNA expression plasmids encoding the indicated myc-ULK1 derivatives were used for transfections and recombinant proteins were immunoprecipitated (c-Myc-IP) from HEK293T cells, inactivated, and subjected to in vitro kinase assays using GST-PKCδ recombinant protein as kinase. ( Top panel ) Autoradiography film demonstrating PKCδ-induced phosphorylation of myc-ULK1s and autophosphorylation of GST-PKCδ, as indicated. ( Bottom panel ) Coomassie Blue staining depicts loading/molecular weight (MW) of myc-ULK1 constructs. EV, myc-tagged empty vector. Kinase assay shown is representative of two independent experiments. Source data are provided as a Source Data file.

Article Snippet: The immunoprecipitated myc-tagged proteins were heat inactivated at 65 °C for 20 min in a sonicating bath and then used as substrates for active recombinant full-length human PKCδ protein (50 ng/reaction; #P64-10G-10; SignalChem) in the presence of γ- 32 P ATP in kinase assays, following the manufacturer’s instructions (SignalChem).

Techniques: Western Blot, Negative Control, Lysis, Immunoprecipitation, Control, In Vitro, Activity Assay, Recombinant, Autoradiography, Phospho-proteomics, Expressing, Kinase Assay, Transfection, Staining, Molecular Weight, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms

doi: 10.1038/s41467-022-29381-7

Figure Lengend Snippet: a U937 cells were transfected with either control siRNA or PKCδ siRNA. 24 h later, cells were starved overnight (cultured in RPMI medium without FBS) and then were either left untreated or were treated with IFNα (10 4 IU/mL) for 10 or 30 min, as indicated. Immunoblotting analysis was performed for the indicated proteins. Blots are representative of three independent experiments. *unspecific band. b , c Bands for the indicated proteins were scanned and quantified for untreated and 10 min IFNα-treated samples by densitometry, using ImageJ software. Quantified data are means ± SEM of pULK1/GAPDH from three independent experiments. Adjusted p -values are reported by two-way ANOVA followed by Tukey’s multiple comparisons test to assess the differences in ULK1 phosphorylation on Ser341 and Ser495 with treatment (untreated vs. IFNα), siRNA (Ctrl siRNA vs. PKCδ siRNA) and their interaction as predictors. arb. units, arbitrary units. d Immunoblotting analysis of p-p38 MAPK in lysates from Ulk1/2 −/− MEFs transfected with myc-tagged empty vector (EV), ULK1 WT, ULK1 S341A or ULK1 S495A plasmids treated with IFNα (10 4 IU/mL) for 10 min, as indicated. Blots are representative of three independent experiments. e – h ULK1 KO KT-1 cells were transfected with myc-tagged empty vector (EV), ULK1 WT, ULK1 S341A or ULK1 S495A plasmids. e Immunoblotting analysis of ULK1 in lysates from ULK1 KO KT-1 transfected cells, as indicated. Blots are representative of three independent experiments. f – h qRT-PCR analysis of ( f ) IFIT1 , ( g ) OAS1 , and ( h ) IFIT3 in ULK1 KO KT-1 transfected cells treated for 6 h with IFNα (5000 IU/mL). Shown is mRNA expression fold change over respective untreated ULK1 KO KT-1 transfected cells. Data are means ± SEM from three independent experiments. Adjusted p -values are reported by one-way ANOVA followed by Tukey’s multiple comparisons test. See also Supplementary Figs. – . Source data are provided as a Source Data file.

Article Snippet: The immunoprecipitated myc-tagged proteins were heat inactivated at 65 °C for 20 min in a sonicating bath and then used as substrates for active recombinant full-length human PKCδ protein (50 ng/reaction; #P64-10G-10; SignalChem) in the presence of γ- 32 P ATP in kinase assays, following the manufacturer’s instructions (SignalChem).

Techniques: Transfection, Control, Cell Culture, Western Blot, Software, Phospho-proteomics, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms

doi: 10.1038/s41467-022-29381-7

Figure Lengend Snippet: a Peripheral blood mononuclear cells from patients with PV were transfected with either control siRNA (Ctrl siRNA) or PKCδ siRNA, and the effects of IFNα treatment (10 3 IU/mL) on malignant erythroid (BFU-E) colony formation were assessed by clonogenic assays in methylcellulose. Data are expressed as percent colony formation relative to control siRNA-transfected untreated cells (Ctrl siRNA) and represent means ± SEM of three independent experiments, using cells from three different individual patients with PV. In the graph, data for the same individual patient is represented by the same symbol for each experimental condition. Statistical analyses were performed using a linear mixed effects model, with % colony formation relative to the control group as the outcome, treatment (the three remaining groups) as the fixed effect, and subject as a random effect to account for within-subject correlation between multiple conditions. Kenward-Roger degrees of freedom adjustment was used, which improves performance when sample size is small. Pairwise group comparison tests were adjusted for multiple comparisons using Tukey’s method. Statistical significant p -values (two-sided) are reported. b Correlation between PKCδ ( PRKCD ), ULK1 , or p38 MAPK ( MAPK14 ) baseline mRNA expression and clinical response of PV or ET patients to PEG-IFNα treatment (clinical trial #NCT01259817) based on a simplified, two-group criteria: R, responders ( n = 9); NR, non-responders ( n = 9). The clinical characteristics of each patient are provided in Supplementary Table . 18S gene expression was used as a reference gene. Data are expressed as ΔCt value for each gene, respectively (means ± SEM are shown). Statistical analyses were performed using linear mixed effects models , with Ct as the outcome variable, and target (18S or one of PKCδ, ULK1 or MAPK14 genes), responder status (yes vs. no), and their interaction as fixed effects, and subject as the random effect to account for the correlation between within-subject replicates. Kenward-Roger degrees of freedom adjustment, which improves performance when sample size is small, was used. p -values (two-sided) are reported. Source data are provided as a Source Data file.

Article Snippet: The immunoprecipitated myc-tagged proteins were heat inactivated at 65 °C for 20 min in a sonicating bath and then used as substrates for active recombinant full-length human PKCδ protein (50 ng/reaction; #P64-10G-10; SignalChem) in the presence of γ- 32 P ATP in kinase assays, following the manufacturer’s instructions (SignalChem).

Techniques: Transfection, Control, Comparison, Expressing, Gene Expression

Journal: Nature Communications

Article Title: Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms

doi: 10.1038/s41467-022-29381-7

Figure Lengend Snippet: Type I IFNs bind their transmembrane receptor (IFNAR) activating AKT/mTORC1-dependent phosphorylation of ULK1 on Ser757, which inhibits ULK1 engagement into autophagy-related pathways . In parallel, IFN-mediated downstream activation of PKCδ kinase activity induces ULK1 phosphorylation on Ser341 and Ser495, required for downstream phosphorylation of p38 MAPK and transcription of ISGs, which inhibit proliferation and survival of MPN cells. Engagement of IFNAR also induces activation of caspases leading to apoptosis of MPN cells. However, caspase activation mediates cleavage/activation of ROCK1/2 proteins, found to bind ULK1, that drive pro-survival mechanisms in MPN cells, negatively regulating IFN anti-neoplastic effects.

Article Snippet: The immunoprecipitated myc-tagged proteins were heat inactivated at 65 °C for 20 min in a sonicating bath and then used as substrates for active recombinant full-length human PKCδ protein (50 ng/reaction; #P64-10G-10; SignalChem) in the presence of γ- 32 P ATP in kinase assays, following the manufacturer’s instructions (SignalChem).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay