Journal: The Journal of Biological Chemistry

Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

doi: 10.1074/jbc.RA119.011206

Figure Lengend Snippet: PKCβII is responsible for PMA- and SP600125-induced shedding of Prss14/epithin. A , 427.1.86 cells were pretreated with 5 μ m Go6976 ( Go ) or 1 μ m PKCβ-selective inhibitor (β i ) before treatment with 5 μ m SP600125 ( SP ) or 0.5 μ m PMA for 2 h. B , PKCα or PKCβ knockdown effects on SP600125- or PMA-induced Prss14/epithin shedding. 427.1.86 cells were transfected with 200 n m PKCα or PKCβ siRNA or nontargeting control siRNA for 48 h and then treated with 5 μ m SP600125 or 0.5 μ m of PMA for an additional 2 h. PKCβ knockdown abolished SP600125- and PMA-induced shedding of Prss14/epithin. C , 427.1.86 cells were transfected with 1 μg/ml of the DN form of PKCβI and PKC PKCβII for 72 h. Control cells were transfected with an empty vector. DN forms of PKCβII inhibited shedding. D , PKCβ inhibition of PRSS14 shedding in two human cell lines. PC3 prostate cancer cells and MCF7 breast cancer cells were maintained in serum-free medium overnight and then treated with 1 μ m PKCβ inhibitor for an additional 6 h before testing.

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS, and proteins were detected using specific antibodies: mAb5 , anti PKCβII polyclonal antibody (catalog no. sc-13149, Santa Cruz Biotechnology), anti-phospho-specific PKCβII (Ser-660) polyclonal antibody (catalog no. sc-365463, Santa Cruz Biotechnology), anti-JNK1/2 mAb (catalog no. sc-7345, Santa Cruz Biotechnology), anti-E-cadherin polyclonal antibody (catalog no. sc-8426, Santa Cruz Biotechnology), anti-TACE polyclonal antibody (catalog no. ab13535, Abcam), anti-phospho-specific TACE (Thr-735) polyclonal antibody (catalog no. ab182630, Abcam), and anti-β-actin mAb (catalog no. A1978, Sigma).

Techniques: Knockdown, Transfection, Control, Dominant Negative Mutation, Plasmid Preparation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

doi: 10.1074/jbc.RA119.011206

Figure Lengend Snippet: JNK inhibition increases PKCβII activity, translocation into the membrane, and TACE phosphorylation. A , phosphorylation of PKCβII by the JNK inhibitor. 427.1.86 cells were pretreated with 10 μ m anisomycin ( AN ) for 30 min and then treated with 5 μ m SP600125 ( SP ) for an additional 1 h. SP600125 treatment induced PKCβII phosphorylation. Relative values of band intensity are expressed as means ± S.D. of four independent experiments. ***, p < 0.001. B , kinetics of SP600125- or PMA-induced PKCβII activity. 427.1.86 cells were incubated with 5 μ m SP600125 or 0.5 μ m PMA for 0, 30, 60, and 120 min. After immunoprecipitation with PKCβII antibody, PKCβII activities were determined using the ADP-Glo TM kinase assay kit. All values are expressed as means ± S.D. **, p < 0.01; #, p < 0.05; ##, p < 0.01; n = 3. C , Enzymatic activity of PKCβII was induced by SP600125 alone not by anisomycin combination to SP600125. 427.1.86 cells were pretreated with 1 μ m anisomycin for 30 min and then threated with 5 μ m SP600125 for an additional 1 h. All values are expressed as means ± S.D. **, p < 0.01; n =3. D , immunofluorescent staining of PKCβII-overexpressing 427.1.86 cells. Cells were transfected with 1 μg/ml of PKCβII WT cDNA for 48 h and then treated with 5 μ m SP600125 or 0.5 μ m PMA for 1 h. Immunofluorescence staining was performed with anti PKCβII polyclonal antibody (1:50) followed by FITC-conjugated anti rabbit IgG antibody (1:200). For nucleus staining, cells were incubated with DAPI for 10 min. Membrane localization of PKCβII is indicated by arrows . Images of two cells treated with SP600125 were stylized by embossing the appearance of the signal intensities using Adobe Photoshop. The graph indicates the percentage of cells with PKCβII localized in the membrane from four independent experiments. All values are expressed as means ± S.D. **, p < 0.01; ***, p < 0.001. E , membrane localization of PKCβII by cellular fractionation. 427.1.86 cells were treated with 5 μΜ SP600125 up to 60 min, and then PKCβII in cytosolic and membrane fractions was examined. Relative values of band intensity are expressed as means ± S.D. for three independent experiments. The arrowhead indicates PKCβII. *, p < 0.05. F , phosphorylation of TACE by shedding inducers. In the cells treated with 0.5 μ m PMA or 5 μ m SP600125 for 1 h, phosphorylation of TACE was analyzed by Western blot analysis using an antibody specific for phosphorylated TACE (Thr-735).

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS, and proteins were detected using specific antibodies: mAb5 , anti PKCβII polyclonal antibody (catalog no. sc-13149, Santa Cruz Biotechnology), anti-phospho-specific PKCβII (Ser-660) polyclonal antibody (catalog no. sc-365463, Santa Cruz Biotechnology), anti-JNK1/2 mAb (catalog no. sc-7345, Santa Cruz Biotechnology), anti-E-cadherin polyclonal antibody (catalog no. sc-8426, Santa Cruz Biotechnology), anti-TACE polyclonal antibody (catalog no. ab13535, Abcam), anti-phospho-specific TACE (Thr-735) polyclonal antibody (catalog no. ab182630, Abcam), and anti-β-actin mAb (catalog no. A1978, Sigma).

Techniques: Inhibition, Activity Assay, Translocation Assay, Membrane, Phospho-proteomics, Incubation, Immunoprecipitation, Kinase Assay, Staining, Transfection, Immunofluorescence, Cell Fractionation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

doi: 10.1074/jbc.RA119.011206

Figure Lengend Snippet: PKCβII is critical for PMA- or SP600125-induced cell invasion. A , schematic of the serum induced invasion assay ( left panel ). Invasion of 4271.86 cells depends on TACE activity and Prss14/epithin expression. After serum starvation for 12 h, cells were plated in the upper chamber, and DMSO or 20 μ m TAPI-0 ( TPI ) was added to the lower chamber. Invaded cells on the underside of the membrane after 24 h were stained with crystal violet and counted. The left panel shows the -fold change of the average number of invaded cells in five randomly selected fields. All values are expressed as means ± S.D. *, p < 0.05; **, p < 0.01; n =3. B , PKCβII was knocked down in 427.1.86 cells using PKCβII siRNA ( KD2 , KD3 , and KD4 ). Expression of PKCβII was reduced in KD3 and KD4. C , invasive activity in PMA- and SP600125-treated 427.1.86 and PKCβII knockdown cells. The graph shows the -fold change of the average number of invaded cells on the underside of the membrane after 24 h in the presence or absence of PMA or SP600125. Cells in five different microscopic fields were counted. All statistical analyses were performed using unpaired two-tailed Student's t test. *, p < 0.05; ns , not significant. Error bars , mean ± S.D. ( n = 3). D , model of intracellular signaling events and modulation by SP600125 and PMA during ectodomain shedding of Prss14/epithin.

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS, and proteins were detected using specific antibodies: mAb5 , anti PKCβII polyclonal antibody (catalog no. sc-13149, Santa Cruz Biotechnology), anti-phospho-specific PKCβII (Ser-660) polyclonal antibody (catalog no. sc-365463, Santa Cruz Biotechnology), anti-JNK1/2 mAb (catalog no. sc-7345, Santa Cruz Biotechnology), anti-E-cadherin polyclonal antibody (catalog no. sc-8426, Santa Cruz Biotechnology), anti-TACE polyclonal antibody (catalog no. ab13535, Abcam), anti-phospho-specific TACE (Thr-735) polyclonal antibody (catalog no. ab182630, Abcam), and anti-β-actin mAb (catalog no. A1978, Sigma).

Techniques: Invasion Assay, Activity Assay, Expressing, Membrane, Staining, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

doi: 10.1074/jbc.RA119.011206

Figure Lengend Snippet: PKCβII and JNKs are good prognostic markers in metastatic breast cancer together with ST14. A , box plots presenting mRNA expression of the indicated genes in ER − and ER + breast cancer patients from TCGA datasets. Comparisons were analyzed by unpaired two-tailed Student's t test. B , survival analysis of four breast cancer patient groups divided by expression levels of ST14 and PRKCB ( left panel ) or MAPK9 ( right panel ). p values were calculated using log rank statistics. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns , not significant.

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS, and proteins were detected using specific antibodies: mAb5 , anti PKCβII polyclonal antibody (catalog no. sc-13149, Santa Cruz Biotechnology), anti-phospho-specific PKCβII (Ser-660) polyclonal antibody (catalog no. sc-365463, Santa Cruz Biotechnology), anti-JNK1/2 mAb (catalog no. sc-7345, Santa Cruz Biotechnology), anti-E-cadherin polyclonal antibody (catalog no. sc-8426, Santa Cruz Biotechnology), anti-TACE polyclonal antibody (catalog no. ab13535, Abcam), anti-phospho-specific TACE (Thr-735) polyclonal antibody (catalog no. ab182630, Abcam), and anti-β-actin mAb (catalog no. A1978, Sigma).

Techniques: Expressing, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Classical isoforms of protein kinase C (PKC) and Akt regulate the osteogenic differentiation of human dental follicle cells via both β-catenin and NF-κB

doi: 10.1186/s13287-021-02313-w

Figure Lengend Snippet: Activation of PKC and Akt by Wnt5a and PTHrP. Undifferentiated DFCs were transfected with specific siRNA against WNT5A (Wnt5a, a , b ) or PTHLH (PTHrP, c – e ) or control siRNA for 3 days. Expression of P-PKC (phosphorylation sites according to Ser660 on PKC βII, a , c ), P-Akt (Ser473, b , d ), and NF-κB ( e ) was determined by Western blot analysis. Bar charts show means + SD ( n = 3). Student’s t test was performed to determine statistically significant differences in comparison to control siRNA. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: After taking a photograph of total membrane protein and washing in tris-buffered saline (TBS), membranes were blocked in 5% bovine serum albumin (BSA) or skimmed milk powder (according to dilution of primary antibody) in tris-buffered saline with Tween20 (TBST) for 60 min at room temperature and treated with primary antibodies against PKCα, P-PKC (corresponding to Ser660 on isoform βII), Akt, P-Akt (Ser473), P-SMAD 1/5 (Ser463/465), P-GSK3β (Ser9), NF-κB (p65), P-NF-κB (p65, Ser536), IκBα, IKKα, IKKβ (all diluted 1:1000 in BSA-containing TBST, Cell Signaling Technology, Cambridge, UK), GAPDH (diluted 1:1000 in skimmed milk powder-containing TBST, Cell Signaling Technology), Histone H3 (diluted 1:2000 in BSA-containing TBST, Cell Signaling Technology), or active β-catenin (diluted 1:750 in skimmed milk powder-containing TBST, Merck Millipore) at 4 °C overnight.

Techniques: Activation Assay, Transfection, Expressing, Western Blot