Journal: Molecular Autism

Article Title: Increased expression of the PI3K catalytic subunit p110δ underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family

doi: 10.1186/s13229-015-0066-4

Figure Lengend Snippet: Western blot analyses confirm increased S6 phosphorylation in select SSC cell lines compared to their unaffected siblings. S6 phosphorylation was significantly increased in cell line S1 ( a , n = 3, paired t test, t (2) = 10.7, p = 0.009) and S11 ( b , n = 4, paired t test, t (3) = 10.39, p = 0.002) but not in S3 ( c , n = 4, paired t test, t (3) = 0.72, p = 0.525), confirming ELISA results shown in Fig. . Example Western blots are shown on the top and densitometric quantification of three to four independent experiments below . Phosphorylated S6 was normalized to tubulin. Bar diagrams are means + SEM. None of the class IA PI3K catalytic subunits p110α, p110β, or p110δ was significantly different in S1 or S11 compared to their unaffected siblings (see Additional file : Figure S2)

Article Snippet: The phospho-S6 antibody (Ser235/236, #2211), S6 antibody (#2317), and p110α antibody (#4255) were obtained from Cell Signaling Technology. p110δ antibodies (#sc-7176, mab2687) were obtained from Santa Cruz Biotechnology and R&D Biosciences, respectively.

Techniques: Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation

doi: 10.1042/BCJ20240058

Figure Lengend Snippet: ( A ) Parental HaCaT-TR cells or CARD14 E138A 3xFLAG HaCaT-TR cells were cultured in heavy or light amino acid SILAC media. Cell lysates were mixed according to table and analysed by MS. Scatter plot shows Log 2 ratio of phosphosite intensities, heavy versus light, normalized from forward and reverse SILAC conditions. Red lines = threshold of Log 2 ratio (0.5). Hollow grey circle = detected phosphosite. Annotated phosphosites of interest are highlighted by red squares. ( B ) CARD14 WT -3xFLAG (WT) and CARD14 E138A -3xFLAG (E138A) were induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Cell lysates were immunoblotted for the indicated antigens. ( C ) Expression of CARD14 E138A -3xFLAG was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells (E138A) ± BI605906 (BI6) treatment to block IKK2. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. ( D ) Expression of CARD14 E138A -3xFLAG was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells (E138A) ± AZD8055 (AZD) treatment to block mTOR. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. ( E ) HOIP-deficient (HOIP Def.) and control non-targeted (Con) CARD14 E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 3 h). Cell lysates were immunoblotted. ( F ) TRAF6-deficient (TRAF6 Def.) and control non-targeted (Con) CARD14 E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 3 h). Cell lysates were immunoblotted. ( G ) Expression of CARD14 E138A was induced with tetracycline (Tet; 3 h) in CARD14 E138A 3xFLAG HaCaT-TR cells ± GDC-0941 (GDC) treatment (at indicated concentrations) to inhibit PI 3-kinase. Control cells were treated with DMSO vehicle (Con). Cell lysates were immunoblotted. ( H ) Expression of CARD14 WT -3xFLAG (WT) and CARD14 E138A -3xFLAG (E138A) was induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Confocal imaging determined localization of anti-mTOR and anti-LAMP1 staining. Nuclei were stained with DAPI. Pixel data from three independent experiments were used to determine the degree of signal correlation via Pearson's correlation with Costes method in relevant regions of interest. Means (±SD) are presented and tested by unpaired two tailed t -test to respective control mean for each cell line. ( I ) CARD14 E138A 3xFLAG HaCaT-TR cells were pre-treated with AP2M1 or control (Con) siRNAs. Cell lysates ± tetracycline induction (Tet; 3 h) were immunoblotted for the indicated antigens. Data for S6 kinase phosphorylation from three independent experiments normalized to tubulin loading control are plotted (means ± SD) and analyzed by one-way ANOVA, with correction for multiple testing.

Article Snippet: The following antibodies were used: anti-CARD14 (10400-1-AP; Proteintech), Phospho-S6 Ribosomal Protein (D68F8 Rabbit mAB 5364S; Cell Signalling), S6 Ribosomal Protein (2317; Cell Signalling Technology), anti-actin monoclonal antibody (MP6472J; MP Biomedicals), secondary HRP-conjugated anti-mouse or anti-rabbit IgG antibody (31432 and 31464, respectively; ThermoFisher Scientific).

Techniques: Cell Culture, Multiplex sample analysis, Phospho-proteomics, Expressing, Blocking Assay, Control, Imaging, Staining, Two Tailed Test

Journal: Biochemical Journal

Article Title: CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation

doi: 10.1042/BCJ20240058

Figure Lengend Snippet: ( A ) Expression of CARD11 L232LI -3xFLAG and CARD14 E138A -3xFLAG were induced with tetracycline (Tet; 3 h) in HaCaT-TR cells. Cell lysates were immunoblotted for the indicated antigens. ( B ) Parental HaCaT-TR cells (HaCaT-TR), CARD14 E138A -3xFLAG HaCaT-TR cells (CARD14 E138A ) and CARD11 L232LI -3xFLAG HaCaT-TR cells (CARD11 L232LI ) were induced with tetracycline (Tet; 3 h). Cell lysates were immunoprecipitated with anti-FLAG. Immunoprecipitates (IP) and input lysates were immunoblotted for the indicated antigens. ( C ) Confocal imaging of anti-FLAG localization in CARD14 E138A -3xFLAG HaCaT-TR cells cultured for the indicated times with tetracycline (Tet). Nuclei were stained with DAPI. ( D ) CARD14 E138A -3xFLAG HaCaT-TR cells (CARD14 E138A ) and CARD11 L232LI -3xFLAG HaCaT-TR cells (CARD11 L232LI ) were pre-treated with AP2M1 or control (Con) siRNAs. Cell lysates were prepared after tetracycline induction (Tet; 3 h) and immunoblotted for the indicated antigens. S6 kinase phosphorylation was quantified and normalized to tubulin loading control for three independent experiments. Means (±SD) are presented and tested statistically by unpaired two-tailed t -test relative to control means for each cell line. ( E ) Triplicate cultures of HOIP-deficient (HOIP Def.) and control non-targeted (Con) CARD14 E138A 3xFLAG HaCaT-TR (Cas9) cells were induced for with tetracycline (Tet) for the indicated times. Metabolic activity was monitored by MTT assay (mean ± SD). ( F ) Triplicate cultures of CARD14 E138A 3xFLAG HaCaT-TR cells were induced with tetracycline (Tet; 8 h) plus AZD8055 (AZD) or DMSO vehicle control. Metabolic activity was monitored by MTT assay (mean ± SD). Statistical significance was tested by two tailed, unpaired t -test. ( G ) Triplicate cultures of HOIP-deficient (HOIP Def) and control non-targeted (Con) CARD14 E138A 3xFLAG HaCaT-TR (Cas9) cells were induced with tetracycline (Tet; 6 h). Nascent protein synthesis was monitored by OPP assay (mean ± SD). ( H ) Triplicate cultures of CARD14 E138A 3xFLAG HaCaT-TR cells were induced with tetracycline (Tet; 6 h) plus AZD8055 (AZD) or DMSO vehicle control. Nascent protein synthesis was monitored by OPP assay (mean ± SD). Statistical significance was tested by two tailed, unpaired t -test.

Article Snippet: The following antibodies were used: anti-CARD14 (10400-1-AP; Proteintech), Phospho-S6 Ribosomal Protein (D68F8 Rabbit mAB 5364S; Cell Signalling), S6 Ribosomal Protein (2317; Cell Signalling Technology), anti-actin monoclonal antibody (MP6472J; MP Biomedicals), secondary HRP-conjugated anti-mouse or anti-rabbit IgG antibody (31432 and 31464, respectively; ThermoFisher Scientific).

Techniques: Expressing, Immunoprecipitation, Imaging, Cell Culture, Staining, Control, Phospho-proteomics, Two Tailed Test, Activity Assay, MTT Assay

Journal: Biochemical Journal

Article Title: CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation

doi: 10.1042/BCJ20240058

Figure Lengend Snippet: ( A ) Schematic representation of the experimental procedure for CARD14 E138A induction and rapamycin treatment. CARD14 E138A expression was induced in keratinocytes of Tg CARD14 E138A by daily tamoxifen injections up to 3 days. Rapamycin or control vehicle was injected daily intraperitoneally on d1–d4. Mice were culled on d5 for analysis. Tg control mice were injected with tamoxifen ± rapamycin for comparison. ( B ) Ear sections were produced from mice 5 days after tamoxifen induction ± vehicle (Control) or rapamycin co-injection. Representative sections are shown, stained with hematoxylin and eosin (H&E) and anti-phospho-S6. Scale bar represents 100 µM. ( C ) Skin extracts from ears were produced from mice 5d after tamoxifen induction ± vehicle control (Con) or rapamycin (Rapa) injection and immunoblotted for the indicated antigens. ( D ) Ear sections were produced from mice 5d after tamoxifen induction plus vehicle (Control) or rapamycin treatment. Representative images are shown of ear sections stained with H&E. Scale bar represents 100 µM. ( E ) Changes in ear thickness in mice treated with vehicle (Con) or rapamycin (Rapa) following tamoxifen induction of Tg CARD14 E138A and Tg Control mice. Combined results of two independent experiments are shown. Ear thickness data were analyzed as repeated measurements using the residual maximum likelihood (REML). (Tg Control/vehicle, n = 6; Tg Control/rapamycin n = 7, Tg CARD14 E138A /vehicle, n = 10, Tg CARD14 E138A /rapamycin n = 10). ( F ) Changes in epidermal thickness in Tg CARD14 E138A and Tg Control mice treated with vehicle (Con) or rapamycin (Rapa) following tamoxifen induction. Each symbol represents the mean of at least 10 epidermal thickness measurements for each ear, the line represents the mean value. ( E and F ) Statistical differences were determined using a modified F -test. P values shown. ( G ) Ear sections were produced from Tg CARD14 E138A and Tg Control mice 5d after tamoxifen induction plus vehicle (Control) or rapamycin treatment. Representative images are shown of ear sections stained with H&E and anti-Ki67. Scale bar represents 100 µM. ( H ) Quantification of Ki67 positive cells from control (Tg Control) and Tg CARD14 E138A mice treated with vehicle (Con) or rapamycin (Rapa). Each symbol represents one mouse, the line represents the mean value. Statistical difference between two groups was determined using one-way ANOVA test. * P < 0.01. ( I ) Analysis of infiltrating immune cells in single cell suspension of the ears from Tg Control and Tg CARD14 E138A mice treated or non-treated with rapamycin were analysed by flow cytometry. Cell counts of neutrophils (CD45 + CD3 − CD19 − CD11b + Ly6G + ), DCs (CD45 + CD3/CD19 − CD11b + CD64 − MHCII + CD11c + ), eosinophils (CD45 + CD3/CD19 − CD11b + CD64 − SiglecF + ), T-cells (CD45 + CD3/CD19 + ,MHCII − ). The data was log-transformed and analyzed using a modified F -test. Significant P -values shown. NS, not significant. (Tg Control/vehicle, n = 4; Tg Control/rapamycin, n = 4; Tg CARD14 E138A /vehicle, n = 6; Tg CARD14 E138A /rapamycin, n = 6).

Article Snippet: The following antibodies were used: anti-CARD14 (10400-1-AP; Proteintech), Phospho-S6 Ribosomal Protein (D68F8 Rabbit mAB 5364S; Cell Signalling), S6 Ribosomal Protein (2317; Cell Signalling Technology), anti-actin monoclonal antibody (MP6472J; MP Biomedicals), secondary HRP-conjugated anti-mouse or anti-rabbit IgG antibody (31432 and 31464, respectively; ThermoFisher Scientific).

Techniques: Expressing, Control, Injection, Comparison, Produced, Staining, Modification, Suspension, Flow Cytometry, Transformation Assay

Journal: Cancers

Article Title: A Phase Ib Study of Sotrastaurin, a PKC Inhibitor, and Alpelisib, a PI3Kα Inhibitor, in Patients with Metastatic Uveal Melanoma

doi: 10.3390/cancers13215504

Figure Lengend Snippet: Western blot analysis of PKC and PI3Kα-AKT-mTOR pathway inhibition. Paired tumor biopsies were assessed by Western blot for pAKT, pS6, pERK1/2, pMARCKS, and the respective total proteins. GAPDH was used as a loading control. Clinical outcomes for these respective patients are listed. Protein quantitation of the pre-treatment (left) and post-treatment (right) Western blot analysis performed with Image J. The pre-treatment sample expression level represents a baseline of 100%, with the post-treatment sample expression levels relative to this baseline. Bar plots represent pAKT/total AKT, pS6/total S6, pERK1/2/ total ERK1/2, and pMARCKS/MARCKS, respectively.

Article Snippet: Antibodies used to probe were: pAKT (Ser473, #4060, Clone D9E), Pan AKT (#2920, Clone 40D4), pS6 (S240/244, #4858, Clone D57.2.2E), S6 ribosomal protein (Ser235/236, #2317, Clone 54D2), pMARCKS (Ser152/156, #2741), MARCKS (#5607, Clone D88D11), GAPDH (#5174, Clone D16HI1), pERK1/2 (Y204, #4370, Clone), ERK1/2 (#4695, Clone 137F5), Cyclin D1(#2978, Clone 92G2), Cyclin E1 (#20808, Clone D7T3U), Cyclin A2 (#67955, Clone E6D1J), Bcl-2 (#4223, Clone D55G8), and GLUT4 (Santa Cruz Biotech #SC-53566, Clone IF8), obtained from Cell Signaling Technology Inc., unless otherwise noted.

Techniques: Western Blot, Inhibition, Protein Quantitation, Expressing