card9  (Santa Cruz Biotechnology)

Journal: Journal of Diabetes Investigation

Article Title: Linderalactone mitigates diabetic renal injury by inhibiting macrophage inflammation via the Dectin1/Syk/ CARD9 / IRF5 / NF ‐ κB pathway

doi: 10.1111/jdi.70222

Figure Lengend Snippet: Linderalactone inhibits diabetes‐induced expression and activation of the Dectin1 signaling pathway and inflammatory response in kidney tissues. (a, b) Volcano plot analysis of differentially expressed genes (DEGs) between control group and T1DM group (a) and T1DM and T1DM + LNL (b). (c) Venn diagram of upregulated genes in T1DM compared to control (green), downregulated genes in T1DM + LNL compared to T1DM (orange). (d) Heat map showed mRNA expression of DEGs in kidney tissues derived from control, T1DM, and T1DM + LNL mice based on RNA sequencing. (e, f) Gene set enrichment analysis (GSEA) of the CLR signaling pathway. (g) Immunoblots showed levels of Dectin1, phosphorylated Syk, Syk, CARD9 and IκBα. Nuclear fractions were probed for IRF5 and p65 protein. (h–m) Corresponding densitometric analysis of blots in (g). Values are mean ± SEM (a–f: n = 7 per group; g–m: n = 4 per group).

Article Snippet: IRF5 (1:1000, cat. NO. 96527) and CARD9 (1:1000, cat. NO. 12283) were obtained from CST (MA, USA).

Techniques: Expressing, Activation Assay, Control, Derivative Assay, RNA Sequencing, Western Blot

Journal: Journal of Diabetes Investigation

Article Title: Linderalactone mitigates diabetic renal injury by inhibiting macrophage inflammation via the Dectin1/Syk/ CARD9 / IRF5 / NF ‐ κB pathway

doi: 10.1111/jdi.70222

Figure Lengend Snippet: Linderalactone attenuates HG combining PA‐induced activation of the Dectin1/Syk/CARD9/IRF5/NF‐κB pathway and inflammatory response in RAW264.7 cells. (a–e) Western blot and quantitative analysis of Syk/CARD9/IRF5/NF‐κB signal pathway in HG + PA‐induced RAW264.7 cells. (f) Representative images of immunofluorescence staining for p65 (red) and DAPI (blue). (g–l) Transcript levels of Tnfa (g), Il6 (h), Il1b (i), Il23 (j), Cxcl1 (k), and Csf3 (l) were measured using RT‐qPCR assay in RAW264.7 cells. For f, Scale bar, 50 μm. Values are mean ± SEM (a–e: n = 4 per group; f–l: n = 3 per group).

Article Snippet: IRF5 (1:1000, cat. NO. 96527) and CARD9 (1:1000, cat. NO. 12283) were obtained from CST (MA, USA).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Journal of Diabetes Investigation

Article Title: Linderalactone mitigates diabetic renal injury by inhibiting macrophage inflammation via the Dectin1/Syk/ CARD9 / IRF5 / NF ‐ κB pathway

doi: 10.1111/jdi.70222

Figure Lengend Snippet: Linderalactone inhibits Syk/CARD9/IRF5/NF‐κB pathway activation depending on Dectin1 in RAW264.7 cells. RAW 264.7 cells were transfected with siRNA of Dectin1 or negative control (NC) for 24 h, and then challenged with media containing HG + PA or HG + PA + LNL 20 μM for 1 h. (a–c) Proteins of Syk/CARD9/IRF5/NF‐κB pathway were detected by immunoblotting. (d) Representative images of immunofluorescence staining for p65 (red) and DAPI (blue). RAW264.7 cells were transfected for 24 h with plasmid encoding Dectin1 or empty vector (EV), and stimulated by HG + PA or HG + PA + LNL. (e–g) Protein expression of Syk/CARD9/IRF5/NF‐κB pathway and quantitative analysis. (h) Immunofluorescence staining of p65 and DAPI. (i, j) Transcript mRNA levels of Tnfa , Il6 , and Il1b in RAW264.7 cells transfected with si‐Dectin1 (i) or encoding Dectin1 (j) were measured. Values are mean ± SEM ( n = 3 per group). ns, no significant difference.

Article Snippet: IRF5 (1:1000, cat. NO. 96527) and CARD9 (1:1000, cat. NO. 12283) were obtained from CST (MA, USA).

Techniques: Activation Assay, Transfection, Negative Control, Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Expressing

Journal: mBio

Article Title: Induction of myelopoiesis by Candida dubliniensis drives protective trained immunity against sepsis in a Card9-dependent manner

doi: 10.1128/mbio.02906-25

Figure Lengend Snippet: Card9 is required for protection and bone marrow expansion. ( A ) Experimental design: WT and Myd88 −/− or Card9 −/− C57BL/6 mice were immunized IP with C. dubliniensis (1.75 × 10 7 cells in B or 7 × 10 6 cells in C), followed by lethal IP polymicrobial sepsis challenge (1.75 × 10 7 C. albicans in B or 7 × 10 6 C. albicans in C + 8 × 10 7 S. aureus cells) 14 days later. ( B ) Survival of Myd88 −/− mice following lethal sepsis challenge. n = 9–10/group. ( C ) Survival of Card9 −/− mice following lethal sepsis challenge. n = 15–18/group, combined from two experiments. * P < 0.05, ** P < 0.01, log-rank test with Holm-Sidak multiple comparisons test. ( D ) Experimental design: bone marrow from WT and Card9 −/− C57BL/6 mice was isolated 1–2 days post-IP immunization with 1.75 × 10 7 C. dubliniensis cells. ( E ) C. dubliniensis BM infiltration. n = 5–6/group (WT) and n = 11/group ( Card9 −/− , combined from two experiments). ns , WT vs Card9 −/− at either time point, one-way ANOVA with Sidak’s multiple comparisons test. Data are mean ± s.e.m. ( F ) LKS cell expansion. N = 6–7/group, combined from two experiments. ** P < 0.01, **** P < 0.0001, two-way ANOVA with Sidak’s multiple comparison test. Data are expressed as fold change over naïve mice.

Article Snippet: Female C57BL/6 mice (strain #000664), B6.129- Card9 tm1Xlin /J mice (Card9 KO, strain #028652), and B6.129P2(SJL)- Myd88 tm1.1Defr /J mice (Myd88 null, strain #009088), 5–7 weeks of age, were purchased from Jackson Laboratories.

Techniques: Isolation, Comparison

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: ( A ) expression of recombinant full-length WT-PKCα and mutants D463H and D463N after C3 cleavage. The co-expression of PDK1 kinase domain together with WT-PKCα in insect cells is not necessary to stabilise further the expression of the protein. ( B ) expression of recombinant WT-PKCα and mutants D463H and D463N kinase domains after TEV cleavage of full-length recombinant PKCα. The fraction used for the experiments is highlighted in red. Kinase activities were measured using the ADP-Glo method (C-F). ( C ) Recombinant full-length PKCα activity in vitro on protamine sulphate. The kinase activity of WT is of 523 nmol/min/mg. No activity is detected for both D463 mutants ( D ) Recombinant full-length PKCα activity measured in vitro on a peptide substrate (257 nmol/min/mg on PPSS). There is no unspecific ATPase activity unless at very high saturating PKCα concentration. ( E ) PKCα recombinant isolated kinase domain activity in vitro measured on a peptide substrate. The kinase activity of WT kinase domain is of about 300 nmol/min/mg. No activity is measured for both D463 mutants. ( F ) Co-precipitation of associated PKCα substrates using DARPin pull down of GFP-tagged PKCα WT and mutants expressed in U87MG cells. The substrates ladder is detected by western blot using two different anti phospho-PKCα substrate specific antibodies (pPKCA sub and pPKCA multimab in green). The expression of PKCα is visible (red) using an anti GFP antibody. The phosphorylation of the associated substrates is only detected upon in vitro kinase assay using Mg-ATP, calcium, and lipids activators (phosphatidyl serine). The mutants of the residue D463 do not have kinase activity and do not show specific substrates’ bands phosphorylation. ( G ) Co-precipitation of associated PKCα substrates using DARPin pull down of GFP-tagged WT-PKCα and mutants expressed in U87MG cells. Unlike WT, no substrates’ phosphorylation (WB pPKCA sub) can be detected with the mutants D463H and D463N upon kinase assay.

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Expressing, Recombinant, Activity Assay, In Vitro, Concentration Assay, Isolation, Western Blot, Phospho-proteomics, Kinase Assay, Residue

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: ( A ) U87MG cells expressing transiently GFP-PKCα wild-type and mutants are treated with PMA up to 24 h. Panel (a) the time course decrease in the phosphorylation of the HM motif (pS657) and GFP-PKCα expression upon PMA is detected by western blot with a phospho-specific antibody (green) and an anti-GFP antibody (red). Panel (b) shows the steady state phosphorylation of the three PKCα WT and mutants activation sites (T-loop, TM and HM) prior to PMA stimulation. A one way Anova was used to calculate the significance of the variation between WT and mutants(n=3). Panel (c) shows the decrease in HM phosphorylation upon PMA treatment and an unpaired t-test was used to compare the HM phosphorylation of D463N vs D463H (p-values*) or WT (p-values # ) (n=3). The data in (b) and (c) are expressed as a percentage of decrease relative to the WT signal. ( B )Schematic representation of the life cycle of PKCα upon activation or PMA treatment. PKCα is in an autoinhibited inactive state but in a fully phosphorylated (primed) conformation prior to stimulation. Upon activation the protein translocates to the plasma membrane where the regulatory domains (C1A/C1B and C2) bind to the lipids where PKCα becomes fully active when the pseudo substrate (PSS) is ejected from the catalytic site. The open conformation becomes substrate for the phosphatase that dephosphorylate the priming sites and renders PKCα prone to degradation. ( C ) U87MG cells expressing transiently Myc-PKCα wild-type and mutants are treated with PMA up to 24 h. Panel (a) The time course decrease in the phosphorylation of the turn motif (pT638 TM) and expression of Myc-PKCα upon PMA is detected by western blot with a phospho-specific and anti-Myc antibody. Panel (b) shows the steady state phosphorylation of the three activation sites (T-loop, TM) and HM) of WT-PKCα and mutants prior to PMA. A one way Anova was used to calculate the significance of the variation between WT and D463H or D463N (n=3). Panel (c) shows the decrease in TM phosphorylation upon PMA treatment (n=3). An unpaired t-test was used to compare the TM phosphorylation of D463N vs D463H (p-values*) or WT (p-values # ). The data in panels (b) and (c) are expressed as a percentage of decrease relative to the WT signal. ( D ) Triton-X100 fractionation of U87MG cells expressing Myc-tagged WT-PKCα and mutants following PMA treatment for 4h or 24h (n=3). The soluble and insoluble fractions were analysed by western blot using an anti Myc antibody to calculate the absolute amount of protein upon degradation. An unpaired t-test was used to calculate the significance of the changes between WT and D463H or D463N (p-values*) or D463H vs D463N (p-values # ). ( E ) Soluble and insoluble fractions were also analysed by western blot using an anti pS657 (HM) and the stoichiometry of HM phosphorylation was calculated for each PKCα and mutants. An unpaired t-test was used to calculate the significance of the changes in the soluble and insoluble fractions. In all the experiments the significance was assessed using a t-test P<0.05 (*), P<0.01 (**), P<0.001(***), P<0.0001 (****).

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Expressing, Phospho-proteomics, Western Blot, Activation Assay, Clinical Proteomics, Membrane, Fractionation

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: ( A ) WT and mutant proteins as indicated were expressed as Myc-fusions and captured on anti-MYC beads (described in Materials and Methods). Proteomic analysis of captured proteins was performed by tryptic fragmentation and mass spectrometry and two-way comparisons were made. ( B ) Dendrogram… ( C ) Principal component analysis of the interactors found in each PKCα WT and mutants showing the spatial separation between the Myc-PKC WT and mutants and between the two mutants. STRING analysis on proteins preferentially recovered in D463H immunoprecipitates compared with WT-PKCa immunoprecipitates. ( D ) Heatmap presenting the mutants only interactors (11/17 high confidence hits in D463H only interactors) or interactors found all PKCα or in pair combinations of WT and PKCα mutants. ( E ) STRING network is displayed at 0.15 confidence level demonstrating networking of key epigenetic regulators, including BRD4 and the MLL1 complex components HCFC1 and PRPF31, and their relationship to PKCa (PRKCA). Known interactions from experimentally determined (magenta) or curated datasets (cyan) are indicated, along with associations from text mining (green).

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Mutagenesis, Mass Spectrometry

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: ( A ) mRNA expression of PRKCA in stable U87MG cells with Myc-tagged WT-PKCα, D463H and D463N mutants determined by RNA sequencing. Statistical comparison to parental cells was performed using an unpaired t-test P<0.001 (***) (n=3). ( B ) Stable protein expression of Myc-tagged WT-PKCα and mutants D463H and D463N in stable U87MG by western blot using an anti Myc antibody. ( C ) Principal component analysis of batch-corrected mRNA expression from the different stable cell lines expressing Myc-tagged WT-PKCα and mutants. ( D ) Unsupervised hierarchical clustering analysis of gene expression from all significantly differentially expressed genes in stable U87MG cells with WT-PKCα, D463H and D463N mutants when compared with parental cells. ( E ) Heatmap of genes differentially expressed with overexpression of Myc-PKCα D463H mutant when compared with parental cells. Tiles are coloured by per-gene z-scores across all samples. Rows are clustered by overlap in significant differential gene expression with WT and D463N samples when compared to parental cell lines. Cancer gene consensus (CGC) known cancer genes are annotated for the D463H differentially expressed only cluster. ( F ) and ( G ) Mean log fold change (F) and distribution of log fold change (G) for the WT-PKCα, D463H and D463N mutants when compared with parental cells for genes significantly differentially expressed in WT or D463H mutant and D463H mutant only when compared with parental cells. ( H ) Overrepresentation analysis of the top eight significant gene ontologies from the metabolic process (GO:0008152) gene ontology category child terms for WT-PKCα, D463H and D463N mutant samples. Upregulated differentially expressed genes in stable U87MG cells with WT-PKCα, D463H and D463N mutants when compared with parental cells were used for overrepresentation analysis.

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Expressing, RNA Sequencing, Comparison, Western Blot, Stable Transfection, Gene Expression, Over Expression, Mutagenesis

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: Differential gene-expression induced by WT-PKC α and PKC α D463H. ( A ) Heatmap of genes differentially expressed between PKCα_D463H and WT cells. Right heatmap annotation identifies overlap in significant differentially expressed genes in D463N expressing cells when compared to WT expressing. Known and candidate cancer genes determined by the network of cancer genes (NCG) database of cancer genes are labelled with lines. The significance of the gene differential expression is labelled with the corresponding gene symbol. ( B ) Enrichment plots from GSEA analysis for hallmark Myc pathway and oncogenic TGFβ UP.v1 UP and P53 DN.v1 DN pathways obtained from the molecular signatures database. Ranks were calculated based on the Wald statistic from differential expression results between PKCα D463H and WT cells. Statistical significance was determined using the fGSEA R package. ( C ) Enrichment plot from GSEA analysis for D463H vs WT.

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Gene Expression, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice

doi: 10.1101/2025.05.28.656646

Figure Lengend Snippet: Resistance to BET inhibitors of PKC α D463H mutant expressing cells. ( A, B ) Inhibition of U87MG WT-PKCα, D463H and D463N expressing stable cell lines inhibited with JQ1. ( C, D ) Inhibition of U87MG WT-PKCα, D463H and D463N expressing stable cell lines inhibited with AZD5351. For clarity of visualisation, each cell lines is separately compared with the parental U87MG cell line. Dose response curves represent the mean viability from three independent biological replicates +/- S.D. Significance was assessed using 2-way ANOVA with Tukey’s multiple comparisons. IC50 values (panels B and D) were calculated separately for each biological replicate and data are presented as the mean +/- standard deviation, with significance assessed by one-way ANOVA compared to the parental control. P<0.05 (*), P<0.01 (**), P<0.001), P<0.0001 (****); n=3. ( E ) Schematic of PKCα and D463H mutant kinase-independent signalling. In the basal catalytically autoinhibited state (Regulatory/Catalytic domains interact), PKCa cannot interact with downstream effector(s); for simplicity this state is indicated as a nucleotide free state (apo) although this is not necessarily the case. On allosteric activation triggering an open conformation, the kinase domain can load ATP, and the ATP-bound state is competent to bind downstream effector(s). In some specific cellular contexts for example in tanycytes and chondrocytes, the formation of a PKCa/effector complex drives a proliferative signal which is switched off through kinase activity-dependent effector release (conformation/stability change). In this model, a tumour promoter such as PMA promotes sustained allosteric activation of the kinase pushing the equilibrium towards a higher effector bound steady state. In the case of the loss-of-activity, gain-of-function D463H mutant that still retains an ATP-bound active conformation, the lack of catalytic activity prevents the normal physiological release of the effector (red cross), sustaining complex formation. The maintenance of the stable PKCa D463H-Effector complex triggers a persistent proliferative output (see text for further discussion).

Article Snippet: Constructs were created by InFusion® cloning in vectors cut with EcoRI. pCDNA3-Myc-PKCα was performed by cloning PCR amplified PKCα with an N-terminal Myc tag using the oligos Myc-PKCα_sense and _anti. pBABE puro-Myc-PKCα was done by cloning of PCR amplified Myc-PKCα using the oligos Myc-PKCα_for and _rev. pBABE puro-3xHA-TurboID-PKCα was done by cloning PCR amplified 3xHA-TurboID from the vector Addgene 3xHA-TurboID (#107171) upstream of the 6xGly-PKCα PCR product using the oligos 3xHA-TurboID_for and _rev and 6xGly-PKCα _for and _rev. pTriEX6-GST-PKCα insect cells expression construct was done by cloning the PCR product of full length PKCα into the vector cut with EcoRI-BamHI, using the primers GST-3C PKCα_for and _rev. pTriEX6-GST-PKCα kinase domain was done by introducing a TEV cleavage site (E-N-L-Y-F-Q-|-G/S, cleavage between Q and G/S) in the hinge region just after the residue 321 of full length PKCα in the pTriEX6-GST-PKCα construct.

Techniques: Mutagenesis, Expressing, Inhibition, Stable Transfection, Standard Deviation, Control, Activation Assay, Activity Assay