antibodies pc2 Search Results


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  • 92
    Thermo Fisher anti pc2 antibody
    Primary sequence and processing sites of proSAAS . Prohormone convertase 2 <t>(PC2)</t> participates in the cleavage of proSAAS at residue 209 (arrow 4). The amount of the underlined peptide little-LEN is 25 times higher in the hippocampus of the Wfs1 KO mice as compared to their wild-type littermates, which prompted the measurement of PC2 activity in Wfs1 KO mice.
    Anti Pc2 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti pkd2
    S99 phosphorylation affects cell motility, and not cell proliferation A, B. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and reseeded in Transwell CIM-plate 16 plates (chemotaxis assays, A), or E-plates (proliferation assays, B). After attachment of cells, cell migration or proliferation was continuously monitored in real-time using the xCELLigence RTCA DP instrument. Error bars (grey) represent five experiments. Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). C, D. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and treated with either DMSO (control) or the PKD inhibitor CID755673 (20 μM, during time of the assay) as indicated ( C ), or were additionally transfected with PKD1.KW or <t>PKD2.KW</t> as indicated ( D ). Transwell assays were performed to assess cell migration towards NIH-3T3-conditioned media at 16 hours. In C: Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). Additionally, samples were analyzed by Western blot for expression and activity of LIMK (α-LIMK, α-pT508-LIMK). In D: Protein expression was controlled by Western blot for PAK4 (α-FLAG), PKD1 (α-PKD1), PKD2 (α-PKD2), or β-actin (for loading control). * indicates statistical significance as compared to vector control (first bar), ** indicates statistical significance as compared to wildtype PAK4 (second bar).
    Anti Pkd2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti pc2
    Removal of adherent cues establishes a highly efficient model of PKD cystogenesis a , Still images from Movie 1 showing cyst formation from a PKD organoid in adherent culture. b , Schematic of high-efficiency organoid cystogenesis protocol. c , Representative images of kidney organoids and d , quantification of cyst formation after two weeks of suspension culture (CTRL1 vs. PKD1 -/- , n=3 separate experiments, ± s.e.m., t(3.663)=21.05, p =5.8949×10 -5 ; CTRL2 vs. <t>PKD2</t> -/- , n=4 separate experiments, ± s.e.m., t(5.458)=10.66, p =7.3731×10 -5 ). e , 6-well (3.5 cm) dishes containing PKD or control organoids after 9 months of culture. Scale bars, 100 μm (a-c) and 1 cm (e).
    Anti Pc2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam anti pkd2
    PKD isoform expression in BV-2 cells and PMM. Gene expression of PKD1–3 in a BV-2 and b PMM was analyzed by qPCR and normalized to HPRT. Values are expressed as mean + SD. <t>PKD2</t> expression in BV-2 and PKD1 in PMM was arbitrarily set to 1. n.d. not detectable. c Protein expression of PKD isoforms was determined by western blotting. Samples from the whole brain and cortex were used as controls. One representative blot out of three is shown. β-Tubulin was used as loading control
    Anti Pkd2, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc anti pkd2
    Effect of regorafenib in combination with CRT0066101 on cell growth Human CRC cells (HCT116 (A,B), HCT116 p53 −/− (C,D), RKO (E,F), and HT-29 (G,H)) and normal colon CCD-841 epithelial cells (I,J) were seeded in 96-well plates and then treated with different concentrations of regorafenib and/or PKD inhibitor (CRT0066101) for 3 or 7 days (CCD-841 cells). Cell growth was determined by the WST-1 assay as outlined in the Methods section. (K,L) HCT116 cells were treated with <t>PKD2</t> siRNA complexed with Lipofectamine2000. After 24 hours, culture medium was changed and various concentrations of regorafenib were added. After 72 hours, cell growth was determined by the WST-1 assay as outlined in the Methods section. Data from 3-5 independent experiments were used to calculate the Combination Index (CI) according to the method of Chou and Talalay.
    Anti Pkd2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore rabbit anti pc2
    Prohormone convertase 1/3 (PC1/3) is absent in α cells from control and PGsKO mice. Immunostaining of a control and PGsKO islet for (A) either glucagon (red) or PC1/3 (green) or for (B) either glucagon (red) or <t>PC2</t> (green). In both cases the merged
    Rabbit Anti Pc2, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam mouse anti pkd2
    Prohormone convertase 1/3 (PC1/3) is absent in α cells from control and PGsKO mice. Immunostaining of a control and PGsKO islet for (A) either glucagon (red) or PC1/3 (green) or for (B) either glucagon (red) or <t>PC2</t> (green). In both cases the merged
    Mouse Anti Pkd2, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bethyl rabbit anti pkd2 antibody affinity purified
    Cold plasma treatment is associated with cleavage of HSP90 chaperone and <t>PKD2</t> degradation. ( A – D ) Various cancer cell lines were subjected to treatment with plasma jet (60 s). Twenty-four hours later, cleared lysates were used for western blot analysis in order to determine the HSP90 and PKD2 abundance.
    Rabbit Anti Pkd2 Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology rabbit anti pc2
    The expression of <t>PC2</t> in the pituitary gland of SPF (D, E and F) and conventional (A, B and C) NC/Nga mice by immunohistochemistry (Fig. 4 -1) and by western blot analysis (Fig. 4 -2). The data show a typical experiment from 10 animals. Scale bar = 10 µm. Values are mean ± SD derived from 10 animals. * p
    Rabbit Anti Pc2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam antibodies against pc2
    Proposed pathogenetic mechanism in LSD chondrocytes. Left: In WT chondrocytes, collagen homeostasis is reached through equilibrium between the pool that needs to be secreted and the pool that needs to be degraded. Within this context, fine-tuned mTORC1 activity is particularly important for autophagy regulation, autophagosome biogenesis, AV-lysosome fusion, and, in turn, <t>PC2</t> homeostasis. A key protein complex required for AV-lysosome fusion is represented by Beclin 1–Vps34–UVRAG, a class III–PI3K complex that produces a pool of PI3P required for vesicle fusion. Right: Lysosomal impairment in LSD is responsible for abnormal mTORC1 signaling. Among the autophagy targets of mTORC1, UVRAG activity is particular sensitive to mTORC1 phosphorylation. Thus, in LSD, a stable complex between UVRAG and Rubicon is seen, and insufficient PI3P is produced. This primarily affects AV-lysosome fusion, leading to AV accumulation and a subsequent delay in procollagen trafficking. To overcome the blockage of AV-lysosome fusion and restore proper collagen homeostasis in LSD, therapeutic options can be directed toward either mTORC1 modulation (repression) or pharmacological modulation of the Beclin 1–Vps34 complex (induction).
    Antibodies Against Pc2, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GeneTex anti pc2
    Biochemical characterization of the stably expressing phogrin-CLIP line. ( A ) Cell lysates of the phogrin-CLIP clone were either left untreated (-), treated with endoglycosidase H (E) or peptide:N-glycosidase F (P). Differences in glycosylation were detected by electromobility shift in SDS-PAGE and subsequent probing by Western Blot with antibodies recognizing the CLIP tag. ( B ) Quantitative PCR of gamma tubulin, endogenous phogrin and overexpressed phogrin-CLIP mRNA levels, normalized to beta actin mRNA. ( C ) Cell lysates of non-transfected INS-1 cells and the clone were probed for phogrin-CLIP (left) or endogenous phogrin (right). ( D ) Cell lysates of INS-1 cells and the clone were probed for SG markers <t>PC2,</t> CPE and ChgA. Tubulin serves as a loading control. ( E ) Glucose-stimulated insulin secretion was compared between INS-1 cells and the phogrin-CLIP line. The stimulation index and glucose-stimulated total insulin (GSTI) are shown. For quantitative PCR and static insulin secretion, samples from 5 independent experiments were analyzed. Statistical significance was calculated using the Mann-Whitney-Wilcoxon test with p-values equaling *
    Anti Pc2, supplied by GeneTex, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    WuXi AppTec anti pc2
    SUMO binding via SIM2 is important for <t>Pc2</t> to regulate lineage-specific gene expression in mouse embryonic stem cells. (A) Effects of Pc2 depletion on total cellular protein sumoylation by either SUMO-1 or SUMO-2 in mESCs. Cells were transfected with
    Anti Pc2, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology rabbit polyclonal pc2
    PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for <t>PC2</t> using anti-mouse PC2 rabbit <t>polyclonal</t> serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is > 800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing LAP TULP3 were treated as in A. The total counted cells are > 350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing Flag PC1 HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were > 1,000 per condition with ∼10% cells being Flag positive. PC1 HA -positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2 L703X GFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were > 120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2 L703X GFP. Data represent means ± SD. Ciliary lengths of PC2 L703X GFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were > 240 and > 140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P
    Rabbit Polyclonal Pc2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse anti pc2
    Elevated proinsulin in islets and serum from Pick1 cKO mice. (a–d) Proinsulin and proinsulin/insulin levels from WT and Pick1 cKO serum (a and b, n = 8) and isolated islets (c and d, n = 3, three groups per type of mouse, 10–20 islets per group). (e) Secreted proinsulin level stimulated with 2.8 or 16.7 mM Glc, n = 3. (f–h) mRNA and protein levels of PC1/3, <t>PC2,</t> and CPE from isolated islets of WT and Pick1 cKO mice. Data are represented as mean ± SEM. * p
    Mouse Anti Pc2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology mouse monoclonal anti pkd2 antibody
    Myocyte <t>PKD2</t> knockout attenuates phenylephrine-induced vasoconstriction, but does not alter pressure or angiotensin II-induced vasoconstriction in hindlimb arteries. ( A ) Mean myogenic tone at 80 mmHg illustrating that myogenic tone is similar in third-, fourth-and fifth-order mesenteric arteries and unaltered by PKD2 knockout ( Pkd2 fl/fl : 3 rd n = 4; 4 th n = 5; 5 th n = 4 and Pkd 2 smKO: 3 rd n = 7; 4 th n = 4; 5 th n = 4). ( B ) Mean data for 60 mM K+-induced constriction in first-and second order mesenteric artery rings ( Pkd2 fl/fl n = 5; Pkd 2 smKO n = 6). ( C ) Mean data for phenylephrine-induced vasoconstriction in pressurized, endothelium-denuded 4 th order mesenteric arteries ( Pkd2 fl/fl , n = 3 and Pkd 2 smKO, n = 3). * indicates p
    Mouse Monoclonal Anti Pkd2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bethyl pkd2 antibody
    Protein kinase D (PKD)–2 expression in human gliomas. (A–C) A set of 53 human gliomas was tested for <t>PKD2</t> expression by immunohistochemistry. Staining intensity of glioma cells was defined as low (1), intermediate (2), and high (3) using
    Pkd2 Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pkd2 polyclonal antibody
    Protein kinase D (PKD)–2 expression in human gliomas. (A–C) A set of 53 human gliomas was tested for <t>PKD2</t> expression by immunohistochemistry. Staining intensity of glioma cells was defined as low (1), intermediate (2), and high (3) using
    Pkd2 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Primary sequence and processing sites of proSAAS . Prohormone convertase 2 (PC2) participates in the cleavage of proSAAS at residue 209 (arrow 4). The amount of the underlined peptide little-LEN is 25 times higher in the hippocampus of the Wfs1 KO mice as compared to their wild-type littermates, which prompted the measurement of PC2 activity in Wfs1 KO mice.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice

    doi: 10.3389/fnmol.2015.00045

    Figure Lengend Snippet: Primary sequence and processing sites of proSAAS . Prohormone convertase 2 (PC2) participates in the cleavage of proSAAS at residue 209 (arrow 4). The amount of the underlined peptide little-LEN is 25 times higher in the hippocampus of the Wfs1 KO mice as compared to their wild-type littermates, which prompted the measurement of PC2 activity in Wfs1 KO mice.

    Article Snippet: A commercial anti-PC2 antibody (Thermo-Scientific, dilution 1:200) was used to detect active isoform of PC2 (mass 69 kDa) and proPC2 (mass 72 kDa).

    Techniques: Sequencing, Mouse Assay, Activity Assay

    Prohormone convertase 2 in the hippocampus of Wfs1 mice. (A) PC2 activity is higher in Wfs1 KO mice (149.9 ± 2.3%, n = 8, ∗∗∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice

    doi: 10.3389/fnmol.2015.00045

    Figure Lengend Snippet: Prohormone convertase 2 in the hippocampus of Wfs1 mice. (A) PC2 activity is higher in Wfs1 KO mice (149.9 ± 2.3%, n = 8, ∗∗∗ p

    Article Snippet: A commercial anti-PC2 antibody (Thermo-Scientific, dilution 1:200) was used to detect active isoform of PC2 (mass 69 kDa) and proPC2 (mass 72 kDa).

    Techniques: Mouse Assay, Activity Assay

    A scheme illustrating possible mechanisms of regulation of PC2 by Wfs1 . ProPC2 with its chaperon 7B2 are transported into endoplasmic reticulum (ER), where they form a complex, which exits the ER without PC2 propeptide cleavage. Thereafter 7B2- proPC2 complex enters Golgi complex, where 7B2 is internally cleaved into 21 kDa amino terminal part (7B2-AT) and 31 amino acids long carboxy-terminal part. Then 7B2-AT-proPC2 complex is transported into maturing secretory granules. In the maturing secretory granules PC2 propeptide is cleaved to an active PC2. Wfs1 protein is localized in the membrane of ER and also secretory granules, where it regulates Ca ++ levels and pH. In turn, pH and Ca ++ are known to regulate activity of PC2. In addition, lack of Wfs1 function leads to increased ER stress, which can alter proper folding of proPC2 and 7B2 in the ER.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice

    doi: 10.3389/fnmol.2015.00045

    Figure Lengend Snippet: A scheme illustrating possible mechanisms of regulation of PC2 by Wfs1 . ProPC2 with its chaperon 7B2 are transported into endoplasmic reticulum (ER), where they form a complex, which exits the ER without PC2 propeptide cleavage. Thereafter 7B2- proPC2 complex enters Golgi complex, where 7B2 is internally cleaved into 21 kDa amino terminal part (7B2-AT) and 31 amino acids long carboxy-terminal part. Then 7B2-AT-proPC2 complex is transported into maturing secretory granules. In the maturing secretory granules PC2 propeptide is cleaved to an active PC2. Wfs1 protein is localized in the membrane of ER and also secretory granules, where it regulates Ca ++ levels and pH. In turn, pH and Ca ++ are known to regulate activity of PC2. In addition, lack of Wfs1 function leads to increased ER stress, which can alter proper folding of proPC2 and 7B2 in the ER.

    Article Snippet: A commercial anti-PC2 antibody (Thermo-Scientific, dilution 1:200) was used to detect active isoform of PC2 (mass 69 kDa) and proPC2 (mass 72 kDa).

    Techniques: Activity Assay

    S99 phosphorylation affects cell motility, and not cell proliferation A, B. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and reseeded in Transwell CIM-plate 16 plates (chemotaxis assays, A), or E-plates (proliferation assays, B). After attachment of cells, cell migration or proliferation was continuously monitored in real-time using the xCELLigence RTCA DP instrument. Error bars (grey) represent five experiments. Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). C, D. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and treated with either DMSO (control) or the PKD inhibitor CID755673 (20 μM, during time of the assay) as indicated ( C ), or were additionally transfected with PKD1.KW or PKD2.KW as indicated ( D ). Transwell assays were performed to assess cell migration towards NIH-3T3-conditioned media at 16 hours. In C: Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). Additionally, samples were analyzed by Western blot for expression and activity of LIMK (α-LIMK, α-pT508-LIMK). In D: Protein expression was controlled by Western blot for PAK4 (α-FLAG), PKD1 (α-PKD1), PKD2 (α-PKD2), or β-actin (for loading control). * indicates statistical significance as compared to vector control (first bar), ** indicates statistical significance as compared to wildtype PAK4 (second bar).

    Journal: The Biochemical journal

    Article Title: Protein Kinase D-mediated phosphorylation at serine 99 regulates localization of p21-activated kinase 4

    doi: 10.1042/BJ20130281

    Figure Lengend Snippet: S99 phosphorylation affects cell motility, and not cell proliferation A, B. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and reseeded in Transwell CIM-plate 16 plates (chemotaxis assays, A), or E-plates (proliferation assays, B). After attachment of cells, cell migration or proliferation was continuously monitored in real-time using the xCELLigence RTCA DP instrument. Error bars (grey) represent five experiments. Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). C, D. HeLa cells were transfected with control vector, wildtype PAK4 or PAK4.S99A mutant, and treated with either DMSO (control) or the PKD inhibitor CID755673 (20 μM, during time of the assay) as indicated ( C ), or were additionally transfected with PKD1.KW or PKD2.KW as indicated ( D ). Transwell assays were performed to assess cell migration towards NIH-3T3-conditioned media at 16 hours. In C: Protein expression was controlled by Western blot (α-FLAG for PAK4 expression, α-β-actin for loading control). Additionally, samples were analyzed by Western blot for expression and activity of LIMK (α-LIMK, α-pT508-LIMK). In D: Protein expression was controlled by Western blot for PAK4 (α-FLAG), PKD1 (α-PKD1), PKD2 (α-PKD2), or β-actin (for loading control). * indicates statistical significance as compared to vector control (first bar), ** indicates statistical significance as compared to wildtype PAK4 (second bar).

    Article Snippet: Anti-PKD1 and anti-14-3-3 antibodies were from Santa Cruz (Santa Cruz, CA), anti-PKD3 from Bethyl Laboratories (Montgomery, TX), anti-HA, anti-FLAG (M2) and anti-β-actin from Sigma-Aldrich (St. Louis, MO), anti-PKD2 and anti-HIS from Millipore (Billerica, MA), anti-pMOTIF (PKD substrate antibody), anti-LIMK, anti-PAK4, anti-pS474-PAK4, anti-cofilin, and anti-pS3-cofilin from Cell Signaling Technology (Danvers, MA).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Chemotaxis Assay, Migration, Expressing, Western Blot, Activity Assay

    Active PKD isoforms effectively inhibit cofilin activity and directed cell migration. A , HeLa cells (0.65 × 10 6 cells, 6-cm dish) were co-transfected with cofilin and control vector, constitutively active PKD1, PKD2, or PKD3 as indicated. Lysates

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4 *

    doi: 10.1074/jbc.M111.259424

    Figure Lengend Snippet: Active PKD isoforms effectively inhibit cofilin activity and directed cell migration. A , HeLa cells (0.65 × 10 6 cells, 6-cm dish) were co-transfected with cofilin and control vector, constitutively active PKD1, PKD2, or PKD3 as indicated. Lysates

    Article Snippet: Anti-PKD2 antibody was from Millipore (Billerica, MA).

    Techniques: Activity Assay, Migration, Transfection, Plasmid Preparation

    PKD regulates PAK4 activity in vivo . A , NMuMG cells (3 × 10 5 , 6-cm dish) were co-transfected with FLAG-tagged PAK4 and either vector control or constitutively active PKD1, PKD2, or PKD3, as indicated. PAK4 was immunoprecipitated (α-FLAG)

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4 *

    doi: 10.1074/jbc.M111.259424

    Figure Lengend Snippet: PKD regulates PAK4 activity in vivo . A , NMuMG cells (3 × 10 5 , 6-cm dish) were co-transfected with FLAG-tagged PAK4 and either vector control or constitutively active PKD1, PKD2, or PKD3, as indicated. PAK4 was immunoprecipitated (α-FLAG)

    Article Snippet: Anti-PKD2 antibody was from Millipore (Billerica, MA).

    Techniques: Activity Assay, In Vivo, Transfection, Plasmid Preparation, Immunoprecipitation

    Active PKD isoforms phosphorylate and inactivate SSH1L. A ). B , HeLa cells (0.5 × 10 6 , 6-cm dish) were transfected with tagged, constitutively active alleles of PKD1, PKD2,

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4 *

    doi: 10.1074/jbc.M111.259424

    Figure Lengend Snippet: Active PKD isoforms phosphorylate and inactivate SSH1L. A ). B , HeLa cells (0.5 × 10 6 , 6-cm dish) were transfected with tagged, constitutively active alleles of PKD1, PKD2,

    Article Snippet: Anti-PKD2 antibody was from Millipore (Billerica, MA).

    Techniques: Transfection

    Expression of PKD2 in normal and IPF lung tissues. Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD2 specific antibody at a dilution of 1∶300. A and B: normal lung BECs and AECs (black arrow) as well as macrophages (blue arrows) were stained negative for the PKD2 antibody. Final magnification: ×400. C: in IPF bronchiolar epithelium, PKD2 (red) was expressed in the cytoplasm and nuclei of BECs (pink arrows) and in smooth muscle cells (red arrows). Final magnification: ×400. D: PKD2 (red) was expressed in macrophages (green arrows) but not neutrophils (blue arrows) in IPF lung alveoli. Final magnification: ×400. E–E3: in the fibrotic areas of IPF lung, PKD2 (red) was expressed in the flat (red arrows) and cuboidal (pink arrows) regenerative AECs lining remodeled fibrotic alveolar septa and/or fibroblast foci. It should be noted that alveolar walls grew and expanded towards the regenerative AECs overexpressing PKD2. Strong PKD2 immunoreactivity was also observed in macrophages (green arrows). The regions indicated in panel E (×100) are shown at higher magnification in E1 (×200), E2 (×400), and E3 (×400).

    Journal: PLoS ONE

    Article Title: Protein Kinase D Is Increased and Activated in Lung Epithelial Cells and Macrophages in Idiopathic Pulmonary Fibrosis

    doi: 10.1371/journal.pone.0101983

    Figure Lengend Snippet: Expression of PKD2 in normal and IPF lung tissues. Normal and IPF lung sections were subjected to immunohistochemical analysis by a PKD2 specific antibody at a dilution of 1∶300. A and B: normal lung BECs and AECs (black arrow) as well as macrophages (blue arrows) were stained negative for the PKD2 antibody. Final magnification: ×400. C: in IPF bronchiolar epithelium, PKD2 (red) was expressed in the cytoplasm and nuclei of BECs (pink arrows) and in smooth muscle cells (red arrows). Final magnification: ×400. D: PKD2 (red) was expressed in macrophages (green arrows) but not neutrophils (blue arrows) in IPF lung alveoli. Final magnification: ×400. E–E3: in the fibrotic areas of IPF lung, PKD2 (red) was expressed in the flat (red arrows) and cuboidal (pink arrows) regenerative AECs lining remodeled fibrotic alveolar septa and/or fibroblast foci. It should be noted that alveolar walls grew and expanded towards the regenerative AECs overexpressing PKD2. Strong PKD2 immunoreactivity was also observed in macrophages (green arrows). The regions indicated in panel E (×100) are shown at higher magnification in E1 (×200), E2 (×400), and E3 (×400).

    Article Snippet: PKD2 antibody was from Millipore (Billerica, MA).

    Techniques: Expressing, Immunohistochemistry, Staining

    Removal of adherent cues establishes a highly efficient model of PKD cystogenesis a , Still images from Movie 1 showing cyst formation from a PKD organoid in adherent culture. b , Schematic of high-efficiency organoid cystogenesis protocol. c , Representative images of kidney organoids and d , quantification of cyst formation after two weeks of suspension culture (CTRL1 vs. PKD1 -/- , n=3 separate experiments, ± s.e.m., t(3.663)=21.05, p =5.8949×10 -5 ; CTRL2 vs. PKD2 -/- , n=4 separate experiments, ± s.e.m., t(5.458)=10.66, p =7.3731×10 -5 ). e , 6-well (3.5 cm) dishes containing PKD or control organoids after 9 months of culture. Scale bars, 100 μm (a-c) and 1 cm (e).

    Journal: Nature materials

    Article Title: Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease

    doi: 10.1038/nmat4994

    Figure Lengend Snippet: Removal of adherent cues establishes a highly efficient model of PKD cystogenesis a , Still images from Movie 1 showing cyst formation from a PKD organoid in adherent culture. b , Schematic of high-efficiency organoid cystogenesis protocol. c , Representative images of kidney organoids and d , quantification of cyst formation after two weeks of suspension culture (CTRL1 vs. PKD1 -/- , n=3 separate experiments, ± s.e.m., t(3.663)=21.05, p =5.8949×10 -5 ; CTRL2 vs. PKD2 -/- , n=4 separate experiments, ± s.e.m., t(5.458)=10.66, p =7.3731×10 -5 ). e , 6-well (3.5 cm) dishes containing PKD or control organoids after 9 months of culture. Scale bars, 100 μm (a-c) and 1 cm (e).

    Article Snippet: The antibodies used for the immunoblots were anti-PC1 (sc-130554, Santa Cruz), anti-PC2 (sc-10376, Santa Cruz) and anti-β-actin (4970S, Cell signaling).

    Techniques:

    Outgrowth of PKD cell lines reveals a critical deficiency in PC1 expression. a , Phase contrast image of organoid explants on days 1, 4, and 12 after replating. b , Wide-field fluorescence and c-d , confocal sections showing epithelial and kidney-specific marker expression in representative kidney organoid cell monolayers. e , Representative immunoblots of PC1 and PC2 in kidney organoids and f , undifferentiated hPSCs. g, PC1 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n = 6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.936)=6.603, p =0.00031 (***); ctrl vs. PKD2-/-, t(4.837)=5.669, p =0.0026 (**). h , PC2 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n=6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.451)=0.9247, p =0.3884 (ns); ctrl vs. PKD2-/-, t(5)=8.006, p =0.0005 (***)). i , Representative immunoblot and j-k , quantification of PC1 and PC2 levels in hPSCs treated with four different PKD2 siRNAs (pooled or individually) or a scrambled (scr) siRNA control (n=3). j , Unpaired t test with Welch's correction, scr vs. pool, t(2)=11, p =0.0075; #2 vs. scr, t(2)=1.747, p =0.2227; #3 vs. scr, t(2)=22.66, p =0.0019; #4 vs. scr, t(2)=9.467, p =0.0110; #5 vs. scr, t(2)=11.56, p =0.0074. k , Unpaired t test with Welch's correction, scr vs. pool, t(2)= 16.92, p =0.0035; #2 vs. scr, t(2)=2.912, p =0.1005 (ns, not significant); #3 vs. scr, t(2)=31.93, p =0.0010; #4 vs. scr, t(2)=77.64, p =0.0002; #5 vs. scr, t(2)=20.28, p =0.0024. Scale bars, 100 μm (a-b) or 10 μm (c-d). ns, not significant.

    Journal: Nature materials

    Article Title: Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease

    doi: 10.1038/nmat4994

    Figure Lengend Snippet: Outgrowth of PKD cell lines reveals a critical deficiency in PC1 expression. a , Phase contrast image of organoid explants on days 1, 4, and 12 after replating. b , Wide-field fluorescence and c-d , confocal sections showing epithelial and kidney-specific marker expression in representative kidney organoid cell monolayers. e , Representative immunoblots of PC1 and PC2 in kidney organoids and f , undifferentiated hPSCs. g, PC1 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n = 6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.936)=6.603, p =0.00031 (***); ctrl vs. PKD2-/-, t(4.837)=5.669, p =0.0026 (**). h , PC2 protein levels in undifferentiated hPSCs, normalized to b-actin loading control (ctrl, n=6; PKD1-/- and PKD2-/-, n=3, ± s.e.m., ctrl vs. PKD1-/-, t(6.451)=0.9247, p =0.3884 (ns); ctrl vs. PKD2-/-, t(5)=8.006, p =0.0005 (***)). i , Representative immunoblot and j-k , quantification of PC1 and PC2 levels in hPSCs treated with four different PKD2 siRNAs (pooled or individually) or a scrambled (scr) siRNA control (n=3). j , Unpaired t test with Welch's correction, scr vs. pool, t(2)=11, p =0.0075; #2 vs. scr, t(2)=1.747, p =0.2227; #3 vs. scr, t(2)=22.66, p =0.0019; #4 vs. scr, t(2)=9.467, p =0.0110; #5 vs. scr, t(2)=11.56, p =0.0074. k , Unpaired t test with Welch's correction, scr vs. pool, t(2)= 16.92, p =0.0035; #2 vs. scr, t(2)=2.912, p =0.1005 (ns, not significant); #3 vs. scr, t(2)=31.93, p =0.0010; #4 vs. scr, t(2)=77.64, p =0.0002; #5 vs. scr, t(2)=20.28, p =0.0024. Scale bars, 100 μm (a-b) or 10 μm (c-d). ns, not significant.

    Article Snippet: The antibodies used for the immunoblots were anti-PC1 (sc-130554, Santa Cruz), anti-PC2 (sc-10376, Santa Cruz) and anti-β-actin (4970S, Cell signaling).

    Techniques: Expressing, Fluorescence, Marker, Western Blot

    Inhibition of the lysosome degradation pathway has no effect on PC2 expression in control cells. In MDCK control cells (PC1 uninduced), PC2 expression remained unchanged after treatment with bafilomycin A (50 n m ), with E64 (50 μ m ) and pepstatin

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: Inhibition of the lysosome degradation pathway has no effect on PC2 expression in control cells. In MDCK control cells (PC1 uninduced), PC2 expression remained unchanged after treatment with bafilomycin A (50 n m ), with E64 (50 μ m ) and pepstatin

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Inhibition, Expressing

    PC1 down-regulates PC2 expression. Full-length mPkd1 was stably transfected into MDCK cells using a Flp-In T-REx core kit (Invitrogen). mPkd1 has an N-terminal Halo Tag and a C-terminal FLAG tag. The transfected MDCK cells were grown on Transwell plates

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC1 down-regulates PC2 expression. Full-length mPkd1 was stably transfected into MDCK cells using a Flp-In T-REx core kit (Invitrogen). mPkd1 has an N-terminal Halo Tag and a C-terminal FLAG tag. The transfected MDCK cells were grown on Transwell plates

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, Stable Transfection, Transfection, FLAG-tag

    PC1 regulates PC2 expression in vivo . A , PC2 expression was decreased in wild-type mouse embryos ( Wt , 14.5 days after conception) when compared with Pkd1 knock-out embryos ( ko ). This is a Western blot, and the entire experiment was repeated three times.

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC1 regulates PC2 expression in vivo . A , PC2 expression was decreased in wild-type mouse embryos ( Wt , 14.5 days after conception) when compared with Pkd1 knock-out embryos ( ko ). This is a Western blot, and the entire experiment was repeated three times.

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, In Vivo, Knock-Out, Western Blot

    PC2 co-immunoprecipitates with HDAC6. When PC1 was not induced in MDCK cells, PC2 co-immunoprecipitated with HDAC6 ( left panels ). When PC1 is induced in MDCK cells, co-immunoprecipitation of PC2 with HDAC6 was greatly reduced ( left panels ). PC1 overexpression

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC2 co-immunoprecipitates with HDAC6. When PC1 was not induced in MDCK cells, PC2 co-immunoprecipitated with HDAC6 ( left panels ). When PC1 is induced in MDCK cells, co-immunoprecipitation of PC2 with HDAC6 was greatly reduced ( left panels ). PC1 overexpression

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Immunoprecipitation, Over Expression

    PC1 down-regulates PC2 expression in a dose-dependent manner. A , the tetracycline dose was gradually decreased to decrease PC1 expression. We found that PC1 down-regulates PC2 expression in a dose-dependent manner: the higher the PC1 expression, the lower

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC1 down-regulates PC2 expression in a dose-dependent manner. A , the tetracycline dose was gradually decreased to decrease PC1 expression. We found that PC1 down-regulates PC2 expression in a dose-dependent manner: the higher the PC1 expression, the lower

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing

    The proteasome inhibitor MG132 increases the expression of recombinant PC1 and leads to further down-regulation of PC2 expression in MDCK cells. For this experiment, we used MDCK cells that were stably transfected with an inducible mPkd1 plasmid (see

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: The proteasome inhibitor MG132 increases the expression of recombinant PC1 and leads to further down-regulation of PC2 expression in MDCK cells. For this experiment, we used MDCK cells that were stably transfected with an inducible mPkd1 plasmid (see

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, Recombinant, Stable Transfection, Transfection, Plasmid Preparation

    PC1 regulates PC2 expression in vivo . A , PC2 expression was decreased in wild-type mouse embryos (15.5 days after conception) when compared with Pkd1 Δ CHA /Δ CHA ). This is a Western blot, and the entire experiment was repeated

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC1 regulates PC2 expression in vivo . A , PC2 expression was decreased in wild-type mouse embryos (15.5 days after conception) when compared with Pkd1 Δ CHA /Δ CHA ). This is a Western blot, and the entire experiment was repeated

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, In Vivo, Western Blot

    Expression of mPkd1-R4227X does not affect the PC2-HDAC6 interaction. In the presence or absence of mPkd1-R4227X, PC2 was co-immunoprecipitated ( IP ) with HDAC6 in MDCK cells ( left panels ). Overexpression of mPkd1-R4227X had no effect on HDAC6 expression

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: Expression of mPkd1-R4227X does not affect the PC2-HDAC6 interaction. In the presence or absence of mPkd1-R4227X, PC2 was co-immunoprecipitated ( IP ) with HDAC6 in MDCK cells ( left panels ). Overexpression of mPkd1-R4227X had no effect on HDAC6 expression

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, Immunoprecipitation, Over Expression

    Inhibition of the aggresome degradation pathway with tubacin prevents down-regulation of PC2 when PC1 is expressed. A , tubacin, a specific HDAC6 inhibitor that blocks the aggresome degradation pathway, prevented the down-regulation of PC2 in PC1-induced

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: Inhibition of the aggresome degradation pathway with tubacin prevents down-regulation of PC2 when PC1 is expressed. A , tubacin, a specific HDAC6 inhibitor that blocks the aggresome degradation pathway, prevented the down-regulation of PC2 in PC1-induced

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Inhibition

    The mPkd1-R4227X mutant does not interact with PC2. We tested the mutant in a co-immunoprecipitation assay to confirm that the mPkd1-R4227X protein does not interact with PC2. Full-length mPkd1 was used as a positive control for the co-immunoprecipitation

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: The mPkd1-R4227X mutant does not interact with PC2. We tested the mutant in a co-immunoprecipitation assay to confirm that the mPkd1-R4227X protein does not interact with PC2. Full-length mPkd1 was used as a positive control for the co-immunoprecipitation

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Positive Control, Immunoprecipitation

    The proteasome inhibitor MG132 increases endogenous PC1 expression and decreases PC2 expression in MDCK cells. A , MDCK cells stably transfected with an empty plasmid were treated with MG132 (1 μ m /ml) for 16 h before harvesting, and control cells

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: The proteasome inhibitor MG132 increases endogenous PC1 expression and decreases PC2 expression in MDCK cells. A , MDCK cells stably transfected with an empty plasmid were treated with MG132 (1 μ m /ml) for 16 h before harvesting, and control cells

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation

    PC1 Down-regulates the Expression of PC2

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: PC1 Down-regulates the Expression of PC2

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing

    MG132 increases endogenous PC1 expression and decreases PC2 expression in mIMCD-3 cells. A , mIMCD-3 cells were treated with MG132 (1 μ m /ml) for 16 h. Control cells were treated with vehicle (DMSO). Treatment with MG-132 led to an up-regulation

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: MG132 increases endogenous PC1 expression and decreases PC2 expression in mIMCD-3 cells. A , mIMCD-3 cells were treated with MG132 (1 μ m /ml) for 16 h. Control cells were treated with vehicle (DMSO). Treatment with MG-132 led to an up-regulation

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Expressing

    Inhibition of the aggresome degradation pathway with tubacin has no effect on PC2 expression in control cells. PC2 expression remained unchanged after tubacin treatment in MDCK control cells (PC1 uninduced). Cells were treated with tubacin (10 μ

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: Inhibition of the aggresome degradation pathway with tubacin has no effect on PC2 expression in control cells. PC2 expression remained unchanged after tubacin treatment in MDCK control cells (PC1 uninduced). Cells were treated with tubacin (10 μ

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Inhibition, Expressing

    Inhibition of the lysosome degradation pathway prevents PC1-driven degradation of PC2. A , inhibition of autophagy with bafilomycin A (50 n m ) or E64 (50 μ m ) with pepstatin A (2.5 μ m ) decreased the degradation of PC2 when PC1 was overexpressed

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: Inhibition of the lysosome degradation pathway prevents PC1-driven degradation of PC2. A , inhibition of autophagy with bafilomycin A (50 n m ) or E64 (50 μ m ) with pepstatin A (2.5 μ m ) decreased the degradation of PC2 when PC1 was overexpressed

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Inhibition

    The mPkd1-R4227X mutant does not affect PC2 expression. To determine whether an interaction between PC1 and PC2 is necessary for the regulation of PC2 expression, we generated a plasmid that includes a truncated mouse Pkd1 (mPkd1-R4227X) lacking the last

    Journal: The Journal of Biological Chemistry

    Article Title: Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway *

    doi: 10.1074/jbc.M113.501205

    Figure Lengend Snippet: The mPkd1-R4227X mutant does not affect PC2 expression. To determine whether an interaction between PC1 and PC2 is necessary for the regulation of PC2 expression, we generated a plasmid that includes a truncated mouse Pkd1 (mPkd1-R4227X) lacking the last

    Article Snippet: The anti-PC2 antibody (2 μl) was added to each lysate and allowed to incubate overnight; 50 μl of A/G-agarose beads (Santa Cruz) were then added, and the mixture was incubated with gentle rocking for 4 h at 4 °C.

    Techniques: Mutagenesis, Expressing, Generated, Plasmid Preparation

    Polycystin-1 (PC1), the product of the polycystic kidney disease 1 ( PKD1 ) gene has multiple cleavage products and glycoforms. (A) Schematic showing PC1 and its proteolytic processing steps as well as the position of the termination of the Trun_PC1 splice form (green; 330 kD). The pink bar represents the entire nonproteolytically processed PC1 molecule (Endoglycosidase H [Endo H] sensitive; 550 kD); blue and red bars represent the G protein–coupled receptor autoproteolysis-inducing GAIN/GPS cleaved forms of the molecule: red is the mature Endo H–resistant form (480 kD), and blue is the immature Endo H–sensitive form (ER form 450 kD). LRR, leucine-rich repeat. (B) Western analysis with the N-terminal antibody 7e12 (IgG1 κ ) showing the uncleaved (pink), cleaved mature (red), immature (blue), and the Trunc_PC1 (green) forms of PC1 (4% polyacrylamide gel). Exosomes were prepared from normal deidentified human urine, and the volume of urine used to make the loaded sample is displayed. MEF, mouse embryo fibroblast (WT; two batches 2 and 4; RCTE, renal cortical tubular epithelial). (C) PC1 was immunoprecipitated (IPed) with anti-PC2 (YCE2) and appropriate isotype control (IgG2a κ ) or antipeptide goat polyclonals to the N terminus of the receptor egg jelly (REJ) domain (amino acids SYDPNLEDGDQT , amino acids 2281…2292 EB11002) or extreme C terminus of PC1 (amino acids RTPLRAKNKVHP , amino acids 4288…4299, EB06870) or nonimmune goat IgG and were then probed with 7e12. (D) Western analysis showing that Trunc_PC1 is made in adult human kidney.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Human-Specific Abnormal Alternative Splicing of Wild-Type PKD1 Induces Premature Termination of Polycystin-1

    doi: 10.1681/ASN.2018040442

    Figure Lengend Snippet: Polycystin-1 (PC1), the product of the polycystic kidney disease 1 ( PKD1 ) gene has multiple cleavage products and glycoforms. (A) Schematic showing PC1 and its proteolytic processing steps as well as the position of the termination of the Trun_PC1 splice form (green; 330 kD). The pink bar represents the entire nonproteolytically processed PC1 molecule (Endoglycosidase H [Endo H] sensitive; 550 kD); blue and red bars represent the G protein–coupled receptor autoproteolysis-inducing GAIN/GPS cleaved forms of the molecule: red is the mature Endo H–resistant form (480 kD), and blue is the immature Endo H–sensitive form (ER form 450 kD). LRR, leucine-rich repeat. (B) Western analysis with the N-terminal antibody 7e12 (IgG1 κ ) showing the uncleaved (pink), cleaved mature (red), immature (blue), and the Trunc_PC1 (green) forms of PC1 (4% polyacrylamide gel). Exosomes were prepared from normal deidentified human urine, and the volume of urine used to make the loaded sample is displayed. MEF, mouse embryo fibroblast (WT; two batches 2 and 4; RCTE, renal cortical tubular epithelial). (C) PC1 was immunoprecipitated (IPed) with anti-PC2 (YCE2) and appropriate isotype control (IgG2a κ ) or antipeptide goat polyclonals to the N terminus of the receptor egg jelly (REJ) domain (amino acids SYDPNLEDGDQT , amino acids 2281…2292 EB11002) or extreme C terminus of PC1 (amino acids RTPLRAKNKVHP , amino acids 4288…4299, EB06870) or nonimmune goat IgG and were then probed with 7e12. (D) Western analysis showing that Trunc_PC1 is made in adult human kidney.

    Article Snippet: The PC2 antibody (YCE2) was cognate to amino acids 687…754 (sc-47734, IgG2a κ ; Santa Cruz Biotechnology).

    Techniques: Western Blot, Immunoprecipitation

    PKD2 channels contribute to phenylephrine-induced mesenteric artery depolarization and INa in mesenteric artery myocytes. ( A ) Representative traces of microelectrode impalements illustrating that phenylephrine (PE, 1 µM)-induced depolarization is attenuated in mesenteric arteries of Pkd 2 smKO mice. Scale bars: Y = 10 mV, X = 20 s. ( B ) Mean membrane potential recordings in pressurized (10 and 80 mmHg) mesenteric arteries and in PE at 80 mmHg ( Pkd2 fl/fl : 10 mmHg, n = 13; 80 mmHg, n = 9; 80 mmHg + PE, n = 15. Pkd 2 smKO: 10 mmHg, n = 11; 80 mmHg, n = 12; 80 mmHg + PE, n = 12). *p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: PKD2 channels contribute to phenylephrine-induced mesenteric artery depolarization and INa in mesenteric artery myocytes. ( A ) Representative traces of microelectrode impalements illustrating that phenylephrine (PE, 1 µM)-induced depolarization is attenuated in mesenteric arteries of Pkd 2 smKO mice. Scale bars: Y = 10 mV, X = 20 s. ( B ) Mean membrane potential recordings in pressurized (10 and 80 mmHg) mesenteric arteries and in PE at 80 mmHg ( Pkd2 fl/fl : 10 mmHg, n = 13; 80 mmHg, n = 9; 80 mmHg + PE, n = 15. Pkd 2 smKO: 10 mmHg, n = 11; 80 mmHg, n = 12; 80 mmHg + PE, n = 12). *p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Mouse Assay

    Arterial myocyte PKD2 knockout attenuates vasoconstriction and arterial wall remodeling during hypertension. ( A ) Mean phenylephrine-induced vasoconstriction in pressurized (80 mmHg) mesenteric arteries from angiotensin II-treated mice ( Pkd2 fl/fl , n = 7–8; Pkd 2 smKO, n = 8).

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Arterial myocyte PKD2 knockout attenuates vasoconstriction and arterial wall remodeling during hypertension. ( A ) Mean phenylephrine-induced vasoconstriction in pressurized (80 mmHg) mesenteric arteries from angiotensin II-treated mice ( Pkd2 fl/fl , n = 7–8; Pkd 2 smKO, n = 8).

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Knock-Out, Mouse Assay

    Angiotensin II-induced hypertension is attenuated in Pkd 2 smKO mice. ( A ) Telemetric blood pressure time course showing the development of angiotensin II-induced hypertension in Pkd2 fl/fl (n = 6) and Pkd 2 smKO mice (n = 9). Osmotic minipumps containing either saline or angiotensin II were implanted one day prior to day 0. * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Angiotensin II-induced hypertension is attenuated in Pkd 2 smKO mice. ( A ) Telemetric blood pressure time course showing the development of angiotensin II-induced hypertension in Pkd2 fl/fl (n = 6) and Pkd 2 smKO mice (n = 9). Osmotic minipumps containing either saline or angiotensin II were implanted one day prior to day 0. * indicates p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Mouse Assay

    Pressure-induced vasoconstriction is attenuated in Pkd 2 smKO mouse hindlimb arteries. ( A ) Representative traces illustrating diameter responses to intravascular pressure in gastrocnemius arteries of Pkd2 fl/fl and Pkd 2 smKO mice. ( B ) Mean data for myogenic tone in gastrocnemius arteries ( Pkd2 fl/fl , n = 5; Pkd 2 smKO, n = 6). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Pressure-induced vasoconstriction is attenuated in Pkd 2 smKO mouse hindlimb arteries. ( A ) Representative traces illustrating diameter responses to intravascular pressure in gastrocnemius arteries of Pkd2 fl/fl and Pkd 2 smKO mice. ( B ) Mean data for myogenic tone in gastrocnemius arteries ( Pkd2 fl/fl , n = 5; Pkd 2 smKO, n = 6). * indicates p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Mouse Assay

    Pressure-induced vasoconstriction is unaltered, whereas phenylephrine-induced vasoconstriction is attenuated, in mesenteric arteries of Pkd 2 smKO mice. ( A ) Mean vasoconstriction over a range of pressures in resistance-size mesenteric arteries from Pkd2 fl/fl (n = 7) and Pkd 2 smKO (n = 9) mice. ( B ) Original recordings of concentration-dependent, phenylephrine (PE)-induced contraction in mesenteric artery rings. ( C ) Mean PE-induced contraction ( Pkd2 fl/fl , n = 5; Pkd 2 smKO, n = 6; *p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Pressure-induced vasoconstriction is unaltered, whereas phenylephrine-induced vasoconstriction is attenuated, in mesenteric arteries of Pkd 2 smKO mice. ( A ) Mean vasoconstriction over a range of pressures in resistance-size mesenteric arteries from Pkd2 fl/fl (n = 7) and Pkd 2 smKO (n = 9) mice. ( B ) Original recordings of concentration-dependent, phenylephrine (PE)-induced contraction in mesenteric artery rings. ( C ) Mean PE-induced contraction ( Pkd2 fl/fl , n = 5; Pkd 2 smKO, n = 6; *p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Mouse Assay, Concentration Assay

    PKD2 channels contribute to pressure-induced hindlimb artery depolarization and swelling-activated Na + currents in hindlimb artery myocytes. ( A ) Representative traces of microelectrode impalements under indicated conditions illustrating that pressure-induced depolarization is attenuated in gastrocnemius arteries of Pkd 2 smKO mice. Phenylephrine (PE) = 1 µM. Scale bars: Y = 10 mV, X = 20 s. ( B ) Mean data for membrane potential recordings in pressurized hindlimb arteries in the absence or presence of PE ( Pkd2 fl/fl : 10 mmHg, n = 11; 100 mmHg, n = 10; 100 mmHg + PE, n = 13 and Pkd 2 smKO: 10 mmHg, n = 11; 100 mmHg, n = 10; 100 mmHg + PE, n = 14). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: PKD2 channels contribute to pressure-induced hindlimb artery depolarization and swelling-activated Na + currents in hindlimb artery myocytes. ( A ) Representative traces of microelectrode impalements under indicated conditions illustrating that pressure-induced depolarization is attenuated in gastrocnemius arteries of Pkd 2 smKO mice. Phenylephrine (PE) = 1 µM. Scale bars: Y = 10 mV, X = 20 s. ( B ) Mean data for membrane potential recordings in pressurized hindlimb arteries in the absence or presence of PE ( Pkd2 fl/fl : 10 mmHg, n = 11; 100 mmHg, n = 10; 100 mmHg + PE, n = 13 and Pkd 2 smKO: 10 mmHg, n = 11; 100 mmHg, n = 10; 100 mmHg + PE, n = 14). * indicates p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Mouse Assay

    Activation of Cre recombinase abolishes PKD2 in arterial myocytes of Pkd2 fl/fl :myh11cre/ERT2 mice. ( A ) RT-PCR showing the absence of PKD2 transcript in isolated myocytes from tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( B ) Western blots illustrating the effect of tamoxifen-treatment in Pkd2 fl/fl and Pkd2 fl/fl :myh11-cre/ERT2 mice on PKD2, CaV1.2L (full-length CaV1.2) and CaV1.2S (short CaV1.2) proteins in mesenteric and hindlimb arteries. ( C ) Mean data for proteins in mesenteric arteries of tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice when compared to those in tamoxifen-treated Pkd2 fl/fl mice. n = 4–7. * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Activation of Cre recombinase abolishes PKD2 in arterial myocytes of Pkd2 fl/fl :myh11cre/ERT2 mice. ( A ) RT-PCR showing the absence of PKD2 transcript in isolated myocytes from tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( B ) Western blots illustrating the effect of tamoxifen-treatment in Pkd2 fl/fl and Pkd2 fl/fl :myh11-cre/ERT2 mice on PKD2, CaV1.2L (full-length CaV1.2) and CaV1.2S (short CaV1.2) proteins in mesenteric and hindlimb arteries. ( C ) Mean data for proteins in mesenteric arteries of tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice when compared to those in tamoxifen-treated Pkd2 fl/fl mice. n = 4–7. * indicates p

    Article Snippet: Membranes were blocked with 5% milk and incubated with the following primary antibodies: CaV 1.2 (Neuromab), PKD1 and PKD2 (Santa Cruz, sc-100415), ANO1, TRPC6 and TRPM4 (Abcam) and actin (MilliporeSigma) overnight at 4°C.

    Techniques: Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot

    PKD isoform expression in BV-2 cells and PMM. Gene expression of PKD1–3 in a BV-2 and b PMM was analyzed by qPCR and normalized to HPRT. Values are expressed as mean + SD. PKD2 expression in BV-2 and PKD1 in PMM was arbitrarily set to 1. n.d. not detectable. c Protein expression of PKD isoforms was determined by western blotting. Samples from the whole brain and cortex were used as controls. One representative blot out of three is shown. β-Tubulin was used as loading control

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: PKD isoform expression in BV-2 cells and PMM. Gene expression of PKD1–3 in a BV-2 and b PMM was analyzed by qPCR and normalized to HPRT. Values are expressed as mean + SD. PKD2 expression in BV-2 and PKD1 in PMM was arbitrarily set to 1. n.d. not detectable. c Protein expression of PKD isoforms was determined by western blotting. Samples from the whole brain and cortex were used as controls. One representative blot out of three is shown. β-Tubulin was used as loading control

    Article Snippet: Anti-PKD1 PKD1 and anti-phospho-PKD1 (pPKD1, Ser744/748 ) rabbit antibodies were from Cell Signaling (Beverly, MA, USA), anti-PKD2 and anti- pPKD2 (Ser848 ) rabbit antibodies were from Abcam (Cambridge, UK), and mouse anti- PKD3 antibody was from Santa Cruz (San Diego, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    PKD2 is mainly responsible for a motile PMM phenotype. PMM were cultured in PDL-coated 24-well plates and transduced by shPKD1 and shPKD2. Control vectors expressing scrambled shRNA were used as control. a Silencing efficacy was assessed 72 h post transduction by qPCR. b – d Cells were serum-starved overnight and treated with 0.1% BSA or 1 μM LPA for 24 h. Velocity, accumulated distance, and Euclidean distance were analyzed. e Serum-starved PMM incubated with 0.1% BSA or 1 μM LPA were lysed, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios are normalized to HPRT expression. Results of three separate experiments in triplicate are presented as mean + SD (* p

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: PKD2 is mainly responsible for a motile PMM phenotype. PMM were cultured in PDL-coated 24-well plates and transduced by shPKD1 and shPKD2. Control vectors expressing scrambled shRNA were used as control. a Silencing efficacy was assessed 72 h post transduction by qPCR. b – d Cells were serum-starved overnight and treated with 0.1% BSA or 1 μM LPA for 24 h. Velocity, accumulated distance, and Euclidean distance were analyzed. e Serum-starved PMM incubated with 0.1% BSA or 1 μM LPA were lysed, RNA was isolated and reverse-transcribed, and the gene products indicated were analyzed by qPCR. Expression ratios are normalized to HPRT expression. Results of three separate experiments in triplicate are presented as mean + SD (* p

    Article Snippet: Anti-PKD1 PKD1 and anti-phospho-PKD1 (pPKD1, Ser744/748 ) rabbit antibodies were from Cell Signaling (Beverly, MA, USA), anti-PKD2 and anti- pPKD2 (Ser848 ) rabbit antibodies were from Abcam (Cambridge, UK), and mouse anti- PKD3 antibody was from Santa Cruz (San Diego, CA, USA).

    Techniques: Cell Culture, Expressing, shRNA, Transduction, Real-time Polymerase Chain Reaction, Incubation, Isolation

    Intracellular trafficking of PKD1 and PKD2 in response to LPA. a BV-2 cells were cultured on chamber slides, serum-starved overnight, and incubated in the presence of 0.1% BSA (control) or LPA (1 μM) for 24 h. Cells were fixed, permeabilized, blocked, and stained for tubulin, PKD2, and nuclei (Hoechst). b , c PMM were cultured on chamber slides and serum-starved overnight. Cells were incubated in the presence of 0.1% BSA (control) or LPA (1 μM) for 24 h and stained with tomato lectin, b PKD1 or c PKD2 antibodies, and nuclei (Hoechst). Images were obtained using a Leica confocal microscope. Scale bars = 20 μm

    Journal: Journal of Neuroinflammation

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    doi: 10.1186/s12974-017-1024-1

    Figure Lengend Snippet: Intracellular trafficking of PKD1 and PKD2 in response to LPA. a BV-2 cells were cultured on chamber slides, serum-starved overnight, and incubated in the presence of 0.1% BSA (control) or LPA (1 μM) for 24 h. Cells were fixed, permeabilized, blocked, and stained for tubulin, PKD2, and nuclei (Hoechst). b , c PMM were cultured on chamber slides and serum-starved overnight. Cells were incubated in the presence of 0.1% BSA (control) or LPA (1 μM) for 24 h and stained with tomato lectin, b PKD1 or c PKD2 antibodies, and nuclei (Hoechst). Images were obtained using a Leica confocal microscope. Scale bars = 20 μm

    Article Snippet: Anti-PKD1 PKD1 and anti-phospho-PKD1 (pPKD1, Ser744/748 ) rabbit antibodies were from Cell Signaling (Beverly, MA, USA), anti-PKD2 and anti- pPKD2 (Ser848 ) rabbit antibodies were from Abcam (Cambridge, UK), and mouse anti- PKD3 antibody was from Santa Cruz (San Diego, CA, USA).

    Techniques: Cell Culture, Incubation, Staining, Microscopy

    Agonist-specific activation patterns for PKD1, PKD2 and PKD3 in cardiac fibroblasts

    Journal: Journal of molecular and cellular cardiology

    Article Title: PHOS-TAG SDS-PAGE RESOLVES AGONIST- AND ISOFORM-SPECIFIC ACTIVATION PATTERNS FOR PKD2 AND PKD3 IN CARDIOMYOCYTES AND CARDIAC FIBROBLASTS

    doi: 10.1016/j.yjmcc.2016.08.005

    Figure Lengend Snippet: Agonist-specific activation patterns for PKD1, PKD2 and PKD3 in cardiac fibroblasts

    Article Snippet: PKD2 (Cat. ab57114, a monoclonal antibody raised against residues 1–110 at the N terminus of human PKD2) was from Abcam.

    Techniques: Activation Assay

    Domain structure and phosphorylation sites on PKD1, PKD2, and PKD3

    Journal: Journal of molecular and cellular cardiology

    Article Title: PHOS-TAG SDS-PAGE RESOLVES AGONIST- AND ISOFORM-SPECIFIC ACTIVATION PATTERNS FOR PKD2 AND PKD3 IN CARDIOMYOCYTES AND CARDIAC FIBROBLASTS

    doi: 10.1016/j.yjmcc.2016.08.005

    Figure Lengend Snippet: Domain structure and phosphorylation sites on PKD1, PKD2, and PKD3

    Article Snippet: PKD2 (Cat. ab57114, a monoclonal antibody raised against residues 1–110 at the N terminus of human PKD2) was from Abcam.

    Techniques:

    The Rho inhibitor C3 toxin prevents PKD2 and PKD3 activation by S1P and thrombin, but not by PMA or H 2 O 2 , in cardiac fibroblasts

    Journal: Journal of molecular and cellular cardiology

    Article Title: PHOS-TAG SDS-PAGE RESOLVES AGONIST- AND ISOFORM-SPECIFIC ACTIVATION PATTERNS FOR PKD2 AND PKD3 IN CARDIOMYOCYTES AND CARDIAC FIBROBLASTS

    doi: 10.1016/j.yjmcc.2016.08.005

    Figure Lengend Snippet: The Rho inhibitor C3 toxin prevents PKD2 and PKD3 activation by S1P and thrombin, but not by PMA or H 2 O 2 , in cardiac fibroblasts

    Article Snippet: PKD2 (Cat. ab57114, a monoclonal antibody raised against residues 1–110 at the N terminus of human PKD2) was from Abcam.

    Techniques: Activation Assay

    PKD2 and PKD3 are activated in an agonist-specific manner in cardiomyocytes

    Journal: Journal of molecular and cellular cardiology

    Article Title: PHOS-TAG SDS-PAGE RESOLVES AGONIST- AND ISOFORM-SPECIFIC ACTIVATION PATTERNS FOR PKD2 AND PKD3 IN CARDIOMYOCYTES AND CARDIAC FIBROBLASTS

    doi: 10.1016/j.yjmcc.2016.08.005

    Figure Lengend Snippet: PKD2 and PKD3 are activated in an agonist-specific manner in cardiomyocytes

    Article Snippet: PKD2 (Cat. ab57114, a monoclonal antibody raised against residues 1–110 at the N terminus of human PKD2) was from Abcam.

    Techniques:

    Slit2 FL cleavage plays a role in the proper diffusion of the protein and is dependent on PC2 (A) Western blot detection of Cerulean and Venus in N2a cells transfected with either dual-tagged Slit2 FL or the uncleavable dual-tagged Slit2 FL Δ. An anti-GFP antibody was used, which recognizes Cerulean and Venus, two GFP derived fluorescent proteins. The protein sizes are indicated on the left (unit: kDa). (B) Thick transverse sections of E4 chick spinal cord FP electroporated with dual-tagged Slit2 FL or dual-tagged Slit2 FL Δ (uncleavable form deprived from the cleavage site generating Slit2N and Slit2C fragments). (C) Intensity ratio of the basal compartment over the apical compartment for Cerulean and Venus in FP electroporated with dual-tagged Slit2 FL or dual-tagged Slit2 FL Δ. (D) Western blot detection of Cerulean and Venus in N2a cells transfected with either dual-tagged Slit2 FL or the uncleavable dual-tagged Slit2 FL Δ and treated with PC2 inhibitor CMK. Tubulin is used as a loading control. (E-F) Ratio of the intensity of the Slit2 FL band (E) or the Slit2C band (F) over the intensity of tubulin band. (G) Immunofluorescent labelling of E4 chick spinal cord transverse sections using an antibody targeting PC2 (left panel) and DAPI (right panel). (H) Close-up of the PC2 labelling in the FP. (I) Quantification of the PC2 labelling in the three compartments delineated by yellow dashed lines. (J) Schematic representation of FP glial cells with Slit2 FL, Slit2 fragments, and PC2 distribution as seen in a transverse section (left side) or sagittal section (right side). Data are shown as the mean ± s.d. in (C-H) and Student test has been applied. ns: non-significant, *: p

    Journal: bioRxiv

    Article Title: Iterative inhibition of commissural growth cone exploration, not post-crossing barrier, ensures forward midline navigation through SlitC-PlxnA1 signaling

    doi: 10.1101/2020.04.21.051359

    Figure Lengend Snippet: Slit2 FL cleavage plays a role in the proper diffusion of the protein and is dependent on PC2 (A) Western blot detection of Cerulean and Venus in N2a cells transfected with either dual-tagged Slit2 FL or the uncleavable dual-tagged Slit2 FL Δ. An anti-GFP antibody was used, which recognizes Cerulean and Venus, two GFP derived fluorescent proteins. The protein sizes are indicated on the left (unit: kDa). (B) Thick transverse sections of E4 chick spinal cord FP electroporated with dual-tagged Slit2 FL or dual-tagged Slit2 FL Δ (uncleavable form deprived from the cleavage site generating Slit2N and Slit2C fragments). (C) Intensity ratio of the basal compartment over the apical compartment for Cerulean and Venus in FP electroporated with dual-tagged Slit2 FL or dual-tagged Slit2 FL Δ. (D) Western blot detection of Cerulean and Venus in N2a cells transfected with either dual-tagged Slit2 FL or the uncleavable dual-tagged Slit2 FL Δ and treated with PC2 inhibitor CMK. Tubulin is used as a loading control. (E-F) Ratio of the intensity of the Slit2 FL band (E) or the Slit2C band (F) over the intensity of tubulin band. (G) Immunofluorescent labelling of E4 chick spinal cord transverse sections using an antibody targeting PC2 (left panel) and DAPI (right panel). (H) Close-up of the PC2 labelling in the FP. (I) Quantification of the PC2 labelling in the three compartments delineated by yellow dashed lines. (J) Schematic representation of FP glial cells with Slit2 FL, Slit2 fragments, and PC2 distribution as seen in a transverse section (left side) or sagittal section (right side). Data are shown as the mean ± s.d. in (C-H) and Student test has been applied. ns: non-significant, *: p

    Article Snippet: Sections were incubated overnight at room temperature with anti-PlxnA1 antibody (gift from Y. Yoshida), anti-Robo3 antibody ((1/100, R & D, AF3076); anti-L1CAM antibody (1:100, A439 Abcam 123990), anti-NgCAM antibody (1:50, 8D9, DSHB), anti-BEN (1:50, BEN, DSHB) or an anti-PC2 antibody (1:100, 3533, Abcam) in 1% BSA diluted in PBS.

    Techniques: Diffusion-based Assay, Western Blot, Transfection, Derivative Assay

    PKD2 knockdown inhibited bombesin-stimulated proliferation of HNSCC cells. a PKD2 depletion suppressed GRP-induced ERK1/2 activation. 686LN cells were transfected with a PKD2-targeting siRNA (si-D2–2) and a non-targeting siRNA (si-NT). Two days after transfection, cells were harvested and analyzed by immunoblotting for the indicated proteins. Representative images from one of four independent experiments are shown. b Knockdown of PKD2 blocked bombesin-induced proliferation of 686LN cells. 686LN cells transfected with si-NT and si-D2–2 were grown in the absence or presence of bombesin (20, 200 nM) for 3 days. Cell proliferation was measured by MTT assay. Representative data from one of three independent experiments are shown. ** P

    Journal: BMC Cancer

    Article Title: Analysis of oncogenic activities of protein kinase D1 in head and neck squamous cell carcinoma

    doi: 10.1186/s12885-018-4965-6

    Figure Lengend Snippet: PKD2 knockdown inhibited bombesin-stimulated proliferation of HNSCC cells. a PKD2 depletion suppressed GRP-induced ERK1/2 activation. 686LN cells were transfected with a PKD2-targeting siRNA (si-D2–2) and a non-targeting siRNA (si-NT). Two days after transfection, cells were harvested and analyzed by immunoblotting for the indicated proteins. Representative images from one of four independent experiments are shown. b Knockdown of PKD2 blocked bombesin-induced proliferation of 686LN cells. 686LN cells transfected with si-NT and si-D2–2 were grown in the absence or presence of bombesin (20, 200 nM) for 3 days. Cell proliferation was measured by MTT assay. Representative data from one of three independent experiments are shown. ** P

    Article Snippet: Primary antibodies used for Western blotting were from the following sources: PKD1, p-S916-PKD1, p-S744/748-PKD1 antibodies were obtained from Cell Signaling Technology (Danvers, MA); p-Ser742-PKD1 antibody was from Invitrogen (Carlsbad, CA); PKD2 antibody was from Abcam (Cambridge, MA); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Enzo (Farmingdale, NY); tubulin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Transfection, MTT Assay

    Effect of regorafenib in combination with CRT0066101 on cell growth Human CRC cells (HCT116 (A,B), HCT116 p53 −/− (C,D), RKO (E,F), and HT-29 (G,H)) and normal colon CCD-841 epithelial cells (I,J) were seeded in 96-well plates and then treated with different concentrations of regorafenib and/or PKD inhibitor (CRT0066101) for 3 or 7 days (CCD-841 cells). Cell growth was determined by the WST-1 assay as outlined in the Methods section. (K,L) HCT116 cells were treated with PKD2 siRNA complexed with Lipofectamine2000. After 24 hours, culture medium was changed and various concentrations of regorafenib were added. After 72 hours, cell growth was determined by the WST-1 assay as outlined in the Methods section. Data from 3-5 independent experiments were used to calculate the Combination Index (CI) according to the method of Chou and Talalay.

    Journal: Oncotarget

    Article Title: The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

    doi:

    Figure Lengend Snippet: Effect of regorafenib in combination with CRT0066101 on cell growth Human CRC cells (HCT116 (A,B), HCT116 p53 −/− (C,D), RKO (E,F), and HT-29 (G,H)) and normal colon CCD-841 epithelial cells (I,J) were seeded in 96-well plates and then treated with different concentrations of regorafenib and/or PKD inhibitor (CRT0066101) for 3 or 7 days (CCD-841 cells). Cell growth was determined by the WST-1 assay as outlined in the Methods section. (K,L) HCT116 cells were treated with PKD2 siRNA complexed with Lipofectamine2000. After 24 hours, culture medium was changed and various concentrations of regorafenib were added. After 72 hours, cell growth was determined by the WST-1 assay as outlined in the Methods section. Data from 3-5 independent experiments were used to calculate the Combination Index (CI) according to the method of Chou and Talalay.

    Article Snippet: The following antibodies were used in the experiments: anti-pTyr204-ERK (#sc-7383; Santa Cruz Biotechnology), anti-ERK (#sc-94; Santa Cruz Biotechnology), anti-pSer473-AKT (#9542; Cell Signaling), anti-AKT (#9272; Cell Signaling), anti-pSer867-PKD2 (#07-385; Upstate), anti-PKD2 (#07-488, Upstate), anti-PARP (#9542; Cell Signaling), anti-p-Ser82-HSP27 (#9709; Cell Signaling), anti-HSP27 (#2402; Cell Signaling), anti-PUMA (#12450; Cell Signaling) and anti-α-tubulin (EMD Biosciences; San Diego, CA).

    Techniques: WST-1 Assay

    Effect of regorafenib and/or PKD inhibitor CRT0066101 on activation of ERK and AKT signaling (A) RKO cells were exposed to regorafenib and/or CRT0066101 for 24 hours, stimulated with 100 nM PMA for 30 minutes, then processed for Western blot analysis of pSer876-PKD2, pS473-AKT, pY204-ERK, total PKD2, AKT and ERK. A representative experiment is shown. (B) HCT116 cells were exposed to regorafenib and CRT0066101 for 24 hours and the level of pSer876-PKD2 and PKD2 protein expression was determined. (C) Values represent the mean relative ratios ± SD from 3-5 experiments. *P

    Journal: Oncotarget

    Article Title: The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

    doi:

    Figure Lengend Snippet: Effect of regorafenib and/or PKD inhibitor CRT0066101 on activation of ERK and AKT signaling (A) RKO cells were exposed to regorafenib and/or CRT0066101 for 24 hours, stimulated with 100 nM PMA for 30 minutes, then processed for Western blot analysis of pSer876-PKD2, pS473-AKT, pY204-ERK, total PKD2, AKT and ERK. A representative experiment is shown. (B) HCT116 cells were exposed to regorafenib and CRT0066101 for 24 hours and the level of pSer876-PKD2 and PKD2 protein expression was determined. (C) Values represent the mean relative ratios ± SD from 3-5 experiments. *P

    Article Snippet: The following antibodies were used in the experiments: anti-pTyr204-ERK (#sc-7383; Santa Cruz Biotechnology), anti-ERK (#sc-94; Santa Cruz Biotechnology), anti-pSer473-AKT (#9542; Cell Signaling), anti-AKT (#9272; Cell Signaling), anti-pSer867-PKD2 (#07-385; Upstate), anti-PKD2 (#07-488, Upstate), anti-PARP (#9542; Cell Signaling), anti-p-Ser82-HSP27 (#9709; Cell Signaling), anti-HSP27 (#2402; Cell Signaling), anti-PUMA (#12450; Cell Signaling) and anti-α-tubulin (EMD Biosciences; San Diego, CA).

    Techniques: Activation Assay, Western Blot, Expressing

    Prohormone convertase 1/3 (PC1/3) is absent in α cells from control and PGsKO mice. Immunostaining of a control and PGsKO islet for (A) either glucagon (red) or PC1/3 (green) or for (B) either glucagon (red) or PC2 (green). In both cases the merged

    Journal: The Journal of endocrinology

    Article Title: Pancreas-specific Gs? deficiency has divergent effects on pancreatic ? and ? cell proliferation

    doi: 10.1677/JOE-10-0030

    Figure Lengend Snippet: Prohormone convertase 1/3 (PC1/3) is absent in α cells from control and PGsKO mice. Immunostaining of a control and PGsKO islet for (A) either glucagon (red) or PC1/3 (green) or for (B) either glucagon (red) or PC2 (green). In both cases the merged

    Article Snippet: Sections were then incubated with mouse anti-insulin (Invitrogen, Carlsbad, CA), rabbit anti-glucagon, rat anti-somastatin (Lab Vision, Fremont, CA), rabbit anti-PC1/3, rabbit anti-PC2 (Millipore) or rabbit anti-Gs α ( ) antibodies, followed by secondary antibodies that were labeled with rhodamine or Cy2 (Jackson ImmunoResearch, West Grove, PA).

    Techniques: Mouse Assay, Immunostaining

    Cold plasma treatment is associated with cleavage of HSP90 chaperone and PKD2 degradation. ( A – D ) Various cancer cell lines were subjected to treatment with plasma jet (60 s). Twenty-four hours later, cleared lysates were used for western blot analysis in order to determine the HSP90 and PKD2 abundance.

    Journal: Scientific Reports

    Article Title: Physical plasma-triggered ROS induces tumor cell death upon cleavage of HSP90 chaperone

    doi: 10.1038/s41598-019-38580-0

    Figure Lengend Snippet: Cold plasma treatment is associated with cleavage of HSP90 chaperone and PKD2 degradation. ( A – D ) Various cancer cell lines were subjected to treatment with plasma jet (60 s). Twenty-four hours later, cleared lysates were used for western blot analysis in order to determine the HSP90 and PKD2 abundance.

    Article Snippet: The following antibodies were used: HSP90β (D-19; Santa Cruz Biotechnology, #sc-1057), PKD2 (Bethyl Laboratories, #A300-073A), cleaved PARP (Cell Signaling, #9542 S), and β-actin (Sigma, #A1978).

    Techniques: Western Blot

    Ectopic PKD2 expression is not sufficient to restore cancer cell viability after treatment with cold plasma. Cancer cells were transduced with a PKD2 expression (PKD2 o.e.) or empty vector (e.v.). After selection with antibiotic, lysates of transduced cancer cells were prepared and SDS-PAGE was conducted with PKD2 antibody. β-actin was used as loading control. ( B,C ) MDA-MB-231 breast and SW480 colon cancer cells (e.v. as well as PKD2 o.e.) were treated with plasma (60 s) and/or 50 nM PU-H71, and cell viability was determined at 24 h.

    Journal: Scientific Reports

    Article Title: Physical plasma-triggered ROS induces tumor cell death upon cleavage of HSP90 chaperone

    doi: 10.1038/s41598-019-38580-0

    Figure Lengend Snippet: Ectopic PKD2 expression is not sufficient to restore cancer cell viability after treatment with cold plasma. Cancer cells were transduced with a PKD2 expression (PKD2 o.e.) or empty vector (e.v.). After selection with antibiotic, lysates of transduced cancer cells were prepared and SDS-PAGE was conducted with PKD2 antibody. β-actin was used as loading control. ( B,C ) MDA-MB-231 breast and SW480 colon cancer cells (e.v. as well as PKD2 o.e.) were treated with plasma (60 s) and/or 50 nM PU-H71, and cell viability was determined at 24 h.

    Article Snippet: The following antibodies were used: HSP90β (D-19; Santa Cruz Biotechnology, #sc-1057), PKD2 (Bethyl Laboratories, #A300-073A), cleaved PARP (Cell Signaling, #9542 S), and β-actin (Sigma, #A1978).

    Techniques: Expressing, Transduction, Plasmid Preparation, Selection, SDS Page, Multiple Displacement Amplification

    Cleavage of HSP90 and degradation of PKD2 following cold plasma treatment is associated with cancer cell death. Physical plasma treatment- generated ROS is followed by HSP90 cleavage and subsequent destabilization and degradation of PKD2. While PKD2 degradation plays an important role in cancer cell death, additional essential molecules such as STK33, also contribute to the apoptotic event. Furthermore, pre-treatment of cancer cells with subliminal doses of HSP90 inhibitor followed by cold plasma treatment boosts cell death in human cancer.

    Journal: Scientific Reports

    Article Title: Physical plasma-triggered ROS induces tumor cell death upon cleavage of HSP90 chaperone

    doi: 10.1038/s41598-019-38580-0

    Figure Lengend Snippet: Cleavage of HSP90 and degradation of PKD2 following cold plasma treatment is associated with cancer cell death. Physical plasma treatment- generated ROS is followed by HSP90 cleavage and subsequent destabilization and degradation of PKD2. While PKD2 degradation plays an important role in cancer cell death, additional essential molecules such as STK33, also contribute to the apoptotic event. Furthermore, pre-treatment of cancer cells with subliminal doses of HSP90 inhibitor followed by cold plasma treatment boosts cell death in human cancer.

    Article Snippet: The following antibodies were used: HSP90β (D-19; Santa Cruz Biotechnology, #sc-1057), PKD2 (Bethyl Laboratories, #A300-073A), cleaved PARP (Cell Signaling, #9542 S), and β-actin (Sigma, #A1978).

    Techniques: Generated

    The expression of PC2 in the pituitary gland of SPF (D, E and F) and conventional (A, B and C) NC/Nga mice by immunohistochemistry (Fig. 4 -1) and by western blot analysis (Fig. 4 -2). The data show a typical experiment from 10 animals. Scale bar = 10 µm. Values are mean ± SD derived from 10 animals. * p

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Mild exercise suppresses exacerbation of dermatitis by increasing cleavage of the ?-endorphin from proopiomelanocortin in NC/Nga mice

    doi: 10.3164/jcbn.12-51

    Figure Lengend Snippet: The expression of PC2 in the pituitary gland of SPF (D, E and F) and conventional (A, B and C) NC/Nga mice by immunohistochemistry (Fig. 4 -1) and by western blot analysis (Fig. 4 -2). The data show a typical experiment from 10 animals. Scale bar = 10 µm. Values are mean ± SD derived from 10 animals. * p

    Article Snippet: The sections of the pituitary gland were washed in PBS and then were subsequently incubated overnight at 4°C with rabbit anti prohormone convertase-1/3 (PC1/3; 1:100) polyclonal antibody (MILLIPORE, Temecula, CA), rabbit anti PC2 (1:100) polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit anti carboxypeptidase E (CPE; 1:100) polyclonal antibody (GeneTex Inc., Irvine, CA), in order to determine the expression of PC1/3, PC2 and CPE.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Western Blot, Derivative Assay

    Proposed pathogenetic mechanism in LSD chondrocytes. Left: In WT chondrocytes, collagen homeostasis is reached through equilibrium between the pool that needs to be secreted and the pool that needs to be degraded. Within this context, fine-tuned mTORC1 activity is particularly important for autophagy regulation, autophagosome biogenesis, AV-lysosome fusion, and, in turn, PC2 homeostasis. A key protein complex required for AV-lysosome fusion is represented by Beclin 1–Vps34–UVRAG, a class III–PI3K complex that produces a pool of PI3P required for vesicle fusion. Right: Lysosomal impairment in LSD is responsible for abnormal mTORC1 signaling. Among the autophagy targets of mTORC1, UVRAG activity is particular sensitive to mTORC1 phosphorylation. Thus, in LSD, a stable complex between UVRAG and Rubicon is seen, and insufficient PI3P is produced. This primarily affects AV-lysosome fusion, leading to AV accumulation and a subsequent delay in procollagen trafficking. To overcome the blockage of AV-lysosome fusion and restore proper collagen homeostasis in LSD, therapeutic options can be directed toward either mTORC1 modulation (repression) or pharmacological modulation of the Beclin 1–Vps34 complex (induction).

    Journal: The Journal of Clinical Investigation

    Article Title: mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

    doi: 10.1172/JCI94130

    Figure Lengend Snippet: Proposed pathogenetic mechanism in LSD chondrocytes. Left: In WT chondrocytes, collagen homeostasis is reached through equilibrium between the pool that needs to be secreted and the pool that needs to be degraded. Within this context, fine-tuned mTORC1 activity is particularly important for autophagy regulation, autophagosome biogenesis, AV-lysosome fusion, and, in turn, PC2 homeostasis. A key protein complex required for AV-lysosome fusion is represented by Beclin 1–Vps34–UVRAG, a class III–PI3K complex that produces a pool of PI3P required for vesicle fusion. Right: Lysosomal impairment in LSD is responsible for abnormal mTORC1 signaling. Among the autophagy targets of mTORC1, UVRAG activity is particular sensitive to mTORC1 phosphorylation. Thus, in LSD, a stable complex between UVRAG and Rubicon is seen, and insufficient PI3P is produced. This primarily affects AV-lysosome fusion, leading to AV accumulation and a subsequent delay in procollagen trafficking. To overcome the blockage of AV-lysosome fusion and restore proper collagen homeostasis in LSD, therapeutic options can be directed toward either mTORC1 modulation (repression) or pharmacological modulation of the Beclin 1–Vps34 complex (induction).

    Article Snippet: Cells were incubated for 1 hour with primary antibodies against PC2 (CII-C1; Hybridoma Bank) and Giantin (ab37266; Abcam).

    Techniques: Activity Assay, Produced

    Altered collagen trafficking and autophagy dysfunction in growth plates of MPSVII mice. ( A ) PC2 secretion was synchronized for 3 hours at 40°C and then shifted to 32°C (ER block release) for the indicated durations (min). Representative images of PC2 localization (red) in ER and Golgi area (Giantin, green) in control chondrocytes and vehicle- or Torin1-treated (6 h, 1 μM) Gusb -KO chondrocytes, 15 and 30 minutes after the ER block release of PC2. Scale bars: 10 μm. ( B ) Quantification of PC2-Giantin colocalization over time. n = 3; n = 50 cells for each time point. ( C ) Representative images of GFP-LC3 puncta (autophagosomes) and Lamp-1 (lysosomes) immunostaining in femoral growth plates from Gusb +/+ GFP-LC3 Tg/+ and Gusb –/– GFP-LC3 Tg/+ mice at P6. Scale bars: 10 μm. ( D ) Quantification of Lamp-1–LC3 colocalization. n = 3 mice per group. ( E ) Representative images of SQSTM1/p62 and Lamp-1 immunofluorescence in femoral growth plates from mice of the indicated genotypes. Scale bars: 10 μm. n = 3 mice per group. Data represent the mean values derived from the indicated number of independent experiments. Error bars indicate the SEM. * P ≤ 0.05, ** P ≤ 0.005, and *** P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test ( B ) and unpaired Student’s t test ( D ).

    Journal: The Journal of Clinical Investigation

    Article Title: mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

    doi: 10.1172/JCI94130

    Figure Lengend Snippet: Altered collagen trafficking and autophagy dysfunction in growth plates of MPSVII mice. ( A ) PC2 secretion was synchronized for 3 hours at 40°C and then shifted to 32°C (ER block release) for the indicated durations (min). Representative images of PC2 localization (red) in ER and Golgi area (Giantin, green) in control chondrocytes and vehicle- or Torin1-treated (6 h, 1 μM) Gusb -KO chondrocytes, 15 and 30 minutes after the ER block release of PC2. Scale bars: 10 μm. ( B ) Quantification of PC2-Giantin colocalization over time. n = 3; n = 50 cells for each time point. ( C ) Representative images of GFP-LC3 puncta (autophagosomes) and Lamp-1 (lysosomes) immunostaining in femoral growth plates from Gusb +/+ GFP-LC3 Tg/+ and Gusb –/– GFP-LC3 Tg/+ mice at P6. Scale bars: 10 μm. ( D ) Quantification of Lamp-1–LC3 colocalization. n = 3 mice per group. ( E ) Representative images of SQSTM1/p62 and Lamp-1 immunofluorescence in femoral growth plates from mice of the indicated genotypes. Scale bars: 10 μm. n = 3 mice per group. Data represent the mean values derived from the indicated number of independent experiments. Error bars indicate the SEM. * P ≤ 0.05, ** P ≤ 0.005, and *** P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test ( B ) and unpaired Student’s t test ( D ).

    Article Snippet: Cells were incubated for 1 hour with primary antibodies against PC2 (CII-C1; Hybridoma Bank) and Giantin (ab37266; Abcam).

    Techniques: Mouse Assay, Blocking Assay, Immunostaining, Immunofluorescence, Derivative Assay

    Normalization of mTORC1 signaling restores collagen trafficking and rescues bone phenotype in MPS VII mice. ( A ) Representative images of femoral growth plate sections isolated from Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice at P15. P-S6 immunostaining (arrows, brown, top row), intracellular PC2 (green, second row), COL X (third row), and COL II (brown, bottom row). Nuclei were counterstained with hematoxylin (pink, top row and bottom row) or DAPI (blue, second row). Scale bars: 100 μm. Insets show magnification ×3. ( B ) Bar graphs show the length of the HZ (COL X + area). n = 5 mice per genotype. ( C ) Quantification of collagen isolated from femoral and tibia cartilages from mice of the indicated genotypes and expressed as the percentage of Gusb +/+ mice. n ≥ 5 mice per genotype. ( D ) Representative images of SQSTM1/p62 and Lamp-1 immunofluorescence in femoral growth plates isolated from Gusb –/– and Gusb –/– Rpt +/– mice at P15. n = 3 mice per group. Scale bars: 10 μm; zoom, ×5. ( E ) Quantification of P62–Lamp-1 colocalization. ( F ) Representative images of Alcian blue and alizarin red staining of femurs and tibiae from P15 Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice. ( G ) Femur and tibia lengths for mice of the indicated genotypes. n = 6 mice per genotype. ( H ) Representative images of Alcian blue and alizarin red staining of femurs and tibiae from P30 Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice. ( I ) Femur and tibia lengths for mice of the indicated genotypes. n = 6 mice per genotype. Data represent the mean values derived from the indicated number of mice. Error bars indicate the SEM. * P ≤ 0.05, ** P ≤ 0.005, and *** P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test ( B , C , G , and I ) and unpaired Student’s t test ( E ).

    Journal: The Journal of Clinical Investigation

    Article Title: mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

    doi: 10.1172/JCI94130

    Figure Lengend Snippet: Normalization of mTORC1 signaling restores collagen trafficking and rescues bone phenotype in MPS VII mice. ( A ) Representative images of femoral growth plate sections isolated from Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice at P15. P-S6 immunostaining (arrows, brown, top row), intracellular PC2 (green, second row), COL X (third row), and COL II (brown, bottom row). Nuclei were counterstained with hematoxylin (pink, top row and bottom row) or DAPI (blue, second row). Scale bars: 100 μm. Insets show magnification ×3. ( B ) Bar graphs show the length of the HZ (COL X + area). n = 5 mice per genotype. ( C ) Quantification of collagen isolated from femoral and tibia cartilages from mice of the indicated genotypes and expressed as the percentage of Gusb +/+ mice. n ≥ 5 mice per genotype. ( D ) Representative images of SQSTM1/p62 and Lamp-1 immunofluorescence in femoral growth plates isolated from Gusb –/– and Gusb –/– Rpt +/– mice at P15. n = 3 mice per group. Scale bars: 10 μm; zoom, ×5. ( E ) Quantification of P62–Lamp-1 colocalization. ( F ) Representative images of Alcian blue and alizarin red staining of femurs and tibiae from P15 Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice. ( G ) Femur and tibia lengths for mice of the indicated genotypes. n = 6 mice per genotype. ( H ) Representative images of Alcian blue and alizarin red staining of femurs and tibiae from P30 Gusb +/+ , Gusb –/– , and Gusb –/– Rpt +/– mice. ( I ) Femur and tibia lengths for mice of the indicated genotypes. n = 6 mice per genotype. Data represent the mean values derived from the indicated number of mice. Error bars indicate the SEM. * P ≤ 0.05, ** P ≤ 0.005, and *** P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test ( B , C , G , and I ) and unpaired Student’s t test ( E ).

    Article Snippet: Cells were incubated for 1 hour with primary antibodies against PC2 (CII-C1; Hybridoma Bank) and Giantin (ab37266; Abcam).

    Techniques: Mouse Assay, Isolation, Immunostaining, Immunofluorescence, Staining, Derivative Assay

    Biochemical characterization of the stably expressing phogrin-CLIP line. ( A ) Cell lysates of the phogrin-CLIP clone were either left untreated (-), treated with endoglycosidase H (E) or peptide:N-glycosidase F (P). Differences in glycosylation were detected by electromobility shift in SDS-PAGE and subsequent probing by Western Blot with antibodies recognizing the CLIP tag. ( B ) Quantitative PCR of gamma tubulin, endogenous phogrin and overexpressed phogrin-CLIP mRNA levels, normalized to beta actin mRNA. ( C ) Cell lysates of non-transfected INS-1 cells and the clone were probed for phogrin-CLIP (left) or endogenous phogrin (right). ( D ) Cell lysates of INS-1 cells and the clone were probed for SG markers PC2, CPE and ChgA. Tubulin serves as a loading control. ( E ) Glucose-stimulated insulin secretion was compared between INS-1 cells and the phogrin-CLIP line. The stimulation index and glucose-stimulated total insulin (GSTI) are shown. For quantitative PCR and static insulin secretion, samples from 5 independent experiments were analyzed. Statistical significance was calculated using the Mann-Whitney-Wilcoxon test with p-values equaling *

    Journal: bioRxiv

    Article Title: Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

    doi: 10.1101/2020.06.03.103770

    Figure Lengend Snippet: Biochemical characterization of the stably expressing phogrin-CLIP line. ( A ) Cell lysates of the phogrin-CLIP clone were either left untreated (-), treated with endoglycosidase H (E) or peptide:N-glycosidase F (P). Differences in glycosylation were detected by electromobility shift in SDS-PAGE and subsequent probing by Western Blot with antibodies recognizing the CLIP tag. ( B ) Quantitative PCR of gamma tubulin, endogenous phogrin and overexpressed phogrin-CLIP mRNA levels, normalized to beta actin mRNA. ( C ) Cell lysates of non-transfected INS-1 cells and the clone were probed for phogrin-CLIP (left) or endogenous phogrin (right). ( D ) Cell lysates of INS-1 cells and the clone were probed for SG markers PC2, CPE and ChgA. Tubulin serves as a loading control. ( E ) Glucose-stimulated insulin secretion was compared between INS-1 cells and the phogrin-CLIP line. The stimulation index and glucose-stimulated total insulin (GSTI) are shown. For quantitative PCR and static insulin secretion, samples from 5 independent experiments were analyzed. Statistical significance was calculated using the Mann-Whitney-Wilcoxon test with p-values equaling *

    Article Snippet: Anti-SNAP was from NEB (P9310S), anti-ChgA from Abcam (ab45179), anti-PDI from Stressgen (SPA-891), anti-GST from Santa Cruz (sc-138), anti-His from Novagen (70796-3), anti-TGN38 from BD Transduction (610898), anti-PC2 from GeneTex (GTX114625), anti-CPE from Sigma (AB5314), anti-gamma-tubulin from Sigma (T-6557), anti-CANX from BD Transduction (610523), anti-ICA69 from Abcam (ab81500), anti-GM130 from BD Transduction (610822) and anti-Syp1 from Synaptic Systems (101011), anti-EEA1 (Thermo Scientific, MA5-14794), anti-LAMP2 (Thermo Scientific, 51-2200).

    Techniques: Stable Transfection, Expressing, Cross-linking Immunoprecipitation, SDS Page, Western Blot, Real-time Polymerase Chain Reaction, Transfection, MANN-WHITNEY

    Characterization of enrichment purity. ( A ) Purified SG lysates were probed for LAMP2, PC2 and Syp1 by Western Blot. ( B ) SG purification with a subsequent second immunodepletion step with antibodies either against LAMP2 alone or in combination with Syp1.

    Journal: bioRxiv

    Article Title: Purification of age-distinct insulin secretory granules through antigen restriction using the CLIP-tag

    doi: 10.1101/2020.06.03.103770

    Figure Lengend Snippet: Characterization of enrichment purity. ( A ) Purified SG lysates were probed for LAMP2, PC2 and Syp1 by Western Blot. ( B ) SG purification with a subsequent second immunodepletion step with antibodies either against LAMP2 alone or in combination with Syp1.

    Article Snippet: Anti-SNAP was from NEB (P9310S), anti-ChgA from Abcam (ab45179), anti-PDI from Stressgen (SPA-891), anti-GST from Santa Cruz (sc-138), anti-His from Novagen (70796-3), anti-TGN38 from BD Transduction (610898), anti-PC2 from GeneTex (GTX114625), anti-CPE from Sigma (AB5314), anti-gamma-tubulin from Sigma (T-6557), anti-CANX from BD Transduction (610523), anti-ICA69 from Abcam (ab81500), anti-GM130 from BD Transduction (610822) and anti-Syp1 from Synaptic Systems (101011), anti-EEA1 (Thermo Scientific, MA5-14794), anti-LAMP2 (Thermo Scientific, 51-2200).

    Techniques: Purification, Western Blot

    SUMO binding via SIM2 is important for Pc2 to regulate lineage-specific gene expression in mouse embryonic stem cells. (A) Effects of Pc2 depletion on total cellular protein sumoylation by either SUMO-1 or SUMO-2 in mESCs. Cells were transfected with

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: SUMO binding via SIM2 is important for Pc2 to regulate lineage-specific gene expression in mouse embryonic stem cells. (A) Effects of Pc2 depletion on total cellular protein sumoylation by either SUMO-1 or SUMO-2 in mESCs. Cells were transfected with

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques: Binding Assay, Expressing, Transfection

    Thioester-bound and non-covalently-associated SUMO in Ubc9 are important for its redistribution by Pc2. (A) Schematic representation of constructs used in biochemical fractionation assays and cell imaging experiments. (B, C, E, F, and G) Fractionation

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: Thioester-bound and non-covalently-associated SUMO in Ubc9 are important for its redistribution by Pc2. (A) Schematic representation of constructs used in biochemical fractionation assays and cell imaging experiments. (B, C, E, F, and G) Fractionation

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques: Construct, Fractionation, Imaging

    In vitro binding between Pc2 and SUMO. (A and B) Nickel affinity pulldown assays of the indicated His-tagged proteins. Schematic diagrams show series of bacterially expressed GST-Pc2 (A) and His 6 -Pc2 (B) fusion proteins used in the in vitro pulldown assays.

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: In vitro binding between Pc2 and SUMO. (A and B) Nickel affinity pulldown assays of the indicated His-tagged proteins. Schematic diagrams show series of bacterially expressed GST-Pc2 (A) and His 6 -Pc2 (B) fusion proteins used in the in vitro pulldown assays.

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques: In Vitro, Binding Assay

    Identification of a SIM in Pc2 by yeast two-hybrid assays. (A) The domain structure of Pc2 is shown schematically: the chromodomain is indicated by a black box, the polyhistidine stretch is indicated by a striped box, and a white box indicates the region

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: Identification of a SIM in Pc2 by yeast two-hybrid assays. (A) The domain structure of Pc2 is shown schematically: the chromodomain is indicated by a black box, the polyhistidine stretch is indicated by a striped box, and a white box indicates the region

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques:

    The SIM2 motif is important for Pc2 autosumoylation and substrate sumoylation. (A) The indicated WT or ΔSIM2 mutant versions of T7-Pc2(1-558) were coexpressed with HA-SUMO-2. Cell lysates were analyzed by immunoblotting (IB) with an anti-T7 antibody

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: The SIM2 motif is important for Pc2 autosumoylation and substrate sumoylation. (A) The indicated WT or ΔSIM2 mutant versions of T7-Pc2(1-558) were coexpressed with HA-SUMO-2. Cell lysates were analyzed by immunoblotting (IB) with an anti-T7 antibody

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques: Mutagenesis

    SIM2 in Pc2 is essential for the subcellular relocalization of Ubc9. (A) Schematic representation of T7-tagged Pc2 constructs used in subcellular fractionation and fluorescent microscopy studies. (B, D, and E) Wild-type, ΔSIM2, or K492R mutant

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: SIM2 in Pc2 is essential for the subcellular relocalization of Ubc9. (A) Schematic representation of T7-tagged Pc2 constructs used in subcellular fractionation and fluorescent microscopy studies. (B, D, and E) Wild-type, ΔSIM2, or K492R mutant

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques: Construct, Fractionation, Microscopy, Mutagenesis

    Model illustrating the role of SIM2 (dark gray rectangle) in Pc2 that coordinates the SUMO machinery-substrate association. Pc2 utilizes its SIM2 motif to coordinate substrate (Sub)-Ubc9 interactions in the context of PcG bodies. The SIM2 motif in Pc2

    Journal: Molecular and Cellular Biology

    Article Title: The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿The SUMO E3 Ligase Activity of Pc2 Is Coordinated through a SUMO Interaction Motif ▿ †

    doi: 10.1128/MCB.01510-09

    Figure Lengend Snippet: Model illustrating the role of SIM2 (dark gray rectangle) in Pc2 that coordinates the SUMO machinery-substrate association. Pc2 utilizes its SIM2 motif to coordinate substrate (Sub)-Ubc9 interactions in the context of PcG bodies. The SIM2 motif in Pc2

    Article Snippet: Western blotting was carried out with Supersignal West Dura Extended Duration substrate (Pierce), as described previously , using the primary antibodies anti-GST, anti-extracellular signal-regulated kinase (anti-ERK), anti-Myc (Santa Cruz), anti-T7 (Novagen), antihemagglutinin (anti-HA) (12CA5; Roche), anti-Flag (Sigma), anti-SUMO-1, anti-SUMO-2 (Biomol), anti-Ubc9 (Santa Cruz), and anti-Pc2 (Abgent).

    Techniques:

    PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for PC2 using anti-mouse PC2 rabbit polyclonal serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is > 800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing LAP TULP3 were treated as in A. The total counted cells are > 350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing Flag PC1 HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were > 1,000 per condition with ∼10% cells being Flag positive. PC1 HA -positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2 L703X GFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were > 120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2 L703X GFP. Data represent means ± SD. Ciliary lengths of PC2 L703X GFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were > 240 and > 140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P

    Journal: The Journal of Cell Biology

    Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

    doi: 10.1083/jcb.201607095

    Figure Lengend Snippet: PC1/2 trafficking to cilia requires Tulp3. (A) mIMCD-K2 cells were sequentially transfected with the indicated 200 nM siRNAs twice for 72 h and serum starved for the last 36 h before fixation and immunostained for PC2 using anti-mouse PC2 rabbit polyclonal serum (from G. Pazour; see the Antibodies section of Materials and methods), acetylated tubulin, and DNA. PC2-positive and -negative cilia are marked by white arrows and arrowheads, respectively. The inset shows PC2 levels in control and Tulp3 siRNA-treated cells by immunoblotting. Data represent means ± SD from three experiments. The total number of cells counted is > 800 per condition. Quantification of PC2 using a separate antibody available commercially in Fig. S5 (A and B). (B) mIMCD-K2 cells expressing LAP TULP3 were treated as in A. The total counted cells are > 350 for each condition. ns, not significant. (C) mIMCD-K2 cells stably expressing Flag PC1 HA were sequentially transfected with the indicated 200-nM siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 36 h before fixation and immunostained for Flag (green), HA (red), AcTub (magenta), and DNA. N-terminal Flag tag allowed determination of PC1-expressing cells. Flag-positive cells were counted for HA-positive cilia from three independent experiments, and total counted cells were > 1,000 per condition with ∼10% cells being Flag positive. PC1 HA -positive and -negative cilia are marked by white arrows and arrowheads, respectively. The magenta arrow marks a cilium in a cell not expressing Flag. Data represent means ± SD. (D) ARPE cells stably expressing PC2 L703X GFP were transfected with the indicated 100 nM siRNAs for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, pericentrin, and DNA. GFP-positive cilia were counted from two experiments, and the total number of GFP-positive cells counted were > 120 per condition. GFP-positive and -negative cilia are marked by white arrows and arrowheads, respectively. Yellow arrows point to perinuclear staining for PC2 L703X GFP. Data represent means ± SD. Ciliary lengths of PC2 L703X GFP-positive cells treated with control and TULP3 siRNA are 8.5 ± 4.6 and 3.2 ± 0.8 µm, respectively. n = 20 each. (E) mIMCD-K2 cells were transfected with indicated siRNAs as in A and immunostained for PC2 (using anti-mouse PC2 rabbit polyclonal serum) or Gpr161, acetylated tubulin, and DNA. Pixel intensities of PC2- and Gpr161-positive cilia are shown to the right. White arrows mark PC2-positive cilia, and yellow arrows point to cilia shown in insets. Total counted cells were > 240 and > 140 per condition for PC2 and Gpr161 immunofluorescence, respectively. (A and C–E) *, P

    Article Snippet: Commercial antibodies used were against α-tubulin (rat YL1/2; Santa Cruz Biotechnology, Inc.), acetylated α-tubulin (mAb 6-11B-1; Sigma-Aldrich; and rabbit polyclonal 5335; Cell Signaling Technology), rabbit polyclonal PC2 (H-280; Santa Cruz Biotechnology, Inc.; Fig. S5, A and B), S tag (mouse monoclonal MAC112; EMD Millipore), Myc tag (goat polyclonal ab9132; Abcam [for immunoblotting and immunofluorescence]), Flag (goat polyclonal ab1257; Abcam [for immunoblotting]; and M2 monoclonal F1804; Sigma-Aldrich [for immunofluorescence]), His tag (mouse monoclonal GT359; Sigma-Aldrich), MBP (mouse monoclonal; New England Biolabs, Inc.), Arl13b (mouse monoclonal N295B/66; NeuroMab), Sstr3 (goat polyclonal sc-11617; Santa Cruz Biotechnology, Inc.), and ACIII (rabbit polyclonal sc-588; Santa Cruz Biotechnology, Inc.).

    Techniques: Transfection, Expressing, Stable Transfection, FLAG-tag, Staining, Immunofluorescence

    Elevated proinsulin in islets and serum from Pick1 cKO mice. (a–d) Proinsulin and proinsulin/insulin levels from WT and Pick1 cKO serum (a and b, n = 8) and isolated islets (c and d, n = 3, three groups per type of mouse, 10–20 islets per group). (e) Secreted proinsulin level stimulated with 2.8 or 16.7 mM Glc, n = 3. (f–h) mRNA and protein levels of PC1/3, PC2, and CPE from isolated islets of WT and Pick1 cKO mice. Data are represented as mean ± SEM. * p

    Journal: Molecular Biology of the Cell

    Article Title: PICK1 is essential for insulin production and the maintenance of glucose homeostasis

    doi: 10.1091/mbc.E17-03-0204

    Figure Lengend Snippet: Elevated proinsulin in islets and serum from Pick1 cKO mice. (a–d) Proinsulin and proinsulin/insulin levels from WT and Pick1 cKO serum (a and b, n = 8) and isolated islets (c and d, n = 3, three groups per type of mouse, 10–20 islets per group). (e) Secreted proinsulin level stimulated with 2.8 or 16.7 mM Glc, n = 3. (f–h) mRNA and protein levels of PC1/3, PC2, and CPE from isolated islets of WT and Pick1 cKO mice. Data are represented as mean ± SEM. * p

    Article Snippet: The mouse anti-PC1/3 (1:1000 for Western blotting) antibody was purchased from Abnova, the mouse anti-PC2 (1:1000 for Western blotting) was purchased from EMD Millipore Corporation, and the mouse anti-CPE (1:2000 for Western blotting) was purchased from BD-Biosciences.

    Techniques: Mouse Assay, Isolation, Gas Chromatography

    Myocyte PKD2 knockout attenuates phenylephrine-induced vasoconstriction, but does not alter pressure or angiotensin II-induced vasoconstriction in hindlimb arteries. ( A ) Mean myogenic tone at 80 mmHg illustrating that myogenic tone is similar in third-, fourth-and fifth-order mesenteric arteries and unaltered by PKD2 knockout ( Pkd2 fl/fl : 3 rd n = 4; 4 th n = 5; 5 th n = 4 and Pkd 2 smKO: 3 rd n = 7; 4 th n = 4; 5 th n = 4). ( B ) Mean data for 60 mM K+-induced constriction in first-and second order mesenteric artery rings ( Pkd2 fl/fl n = 5; Pkd 2 smKO n = 6). ( C ) Mean data for phenylephrine-induced vasoconstriction in pressurized, endothelium-denuded 4 th order mesenteric arteries ( Pkd2 fl/fl , n = 3 and Pkd 2 smKO, n = 3). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Myocyte PKD2 knockout attenuates phenylephrine-induced vasoconstriction, but does not alter pressure or angiotensin II-induced vasoconstriction in hindlimb arteries. ( A ) Mean myogenic tone at 80 mmHg illustrating that myogenic tone is similar in third-, fourth-and fifth-order mesenteric arteries and unaltered by PKD2 knockout ( Pkd2 fl/fl : 3 rd n = 4; 4 th n = 5; 5 th n = 4 and Pkd 2 smKO: 3 rd n = 7; 4 th n = 4; 5 th n = 4). ( B ) Mean data for 60 mM K+-induced constriction in first-and second order mesenteric artery rings ( Pkd2 fl/fl n = 5; Pkd 2 smKO n = 6). ( C ) Mean data for phenylephrine-induced vasoconstriction in pressurized, endothelium-denuded 4 th order mesenteric arteries ( Pkd2 fl/fl , n = 3 and Pkd 2 smKO, n = 3). * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Knock-Out

    PKD2 knockout does not alter swelling-activated Icat in isolated mesenteric artery myocytes. ( A ) Representative I-V relationships from the same isolated mesenteric artery myocytes of Pkd2 fl/fl or Pkd 2 smKO mice in isosmosmotic (300 mOsm), hyposmotic (250 mOsm) and hyposmotic (250 mOsm) + Gd 3+ (100 µM) solutions. ( B ) Mean data for hyposmoticactivated Gd 3+ (100 µM)-sensitive cationic current density at −100 and +100 mV ( Pkd2 fl/fl , n = 6 and Pkd 2 smKO, n = 6).

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: PKD2 knockout does not alter swelling-activated Icat in isolated mesenteric artery myocytes. ( A ) Representative I-V relationships from the same isolated mesenteric artery myocytes of Pkd2 fl/fl or Pkd 2 smKO mice in isosmosmotic (300 mOsm), hyposmotic (250 mOsm) and hyposmotic (250 mOsm) + Gd 3+ (100 µM) solutions. ( B ) Mean data for hyposmoticactivated Gd 3+ (100 µM)-sensitive cationic current density at −100 and +100 mV ( Pkd2 fl/fl , n = 6 and Pkd 2 smKO, n = 6).

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Knock-Out, Isolation, Mouse Assay

    Angiotensin II-induced hypertension is attenuated in Pkd 2 smKO mice. ( A ) Telemetric blood pressure time course showing the development of angiotensin II-induced hypertension in Pkd2 fl/fl (n = 6) and Pkd 2 smKO mice (n = 9). Osmotic minipumps containing either saline or angiotensin II were implanted one day prior to day 0. * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Angiotensin II-induced hypertension is attenuated in Pkd 2 smKO mice. ( A ) Telemetric blood pressure time course showing the development of angiotensin II-induced hypertension in Pkd2 fl/fl (n = 6) and Pkd 2 smKO mice (n = 9). Osmotic minipumps containing either saline or angiotensin II were implanted one day prior to day 0. * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Mouse Assay

    Several proteins that regulate arterial contractility are unchanged in tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( A ) Western blots illustrating Angiotensin II type one receptor (AT1R), Piezo1, α1-adrenergic receptor A (α1A), α1-adrenergic receptor B (α1B), α1-adrenergic receptor D (α1D) and G protein-coupled receptor 68 (GPR68) protein levels in mesenteric and hindlimb arteries of Pkd2 fl/fl and Pkd2 fl/fl :myh11-cre/ERT2 mice. ( B ) Mean data from mesenteric and hindlimb arteries (n = 4 per group).

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Several proteins that regulate arterial contractility are unchanged in tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( A ) Western blots illustrating Angiotensin II type one receptor (AT1R), Piezo1, α1-adrenergic receptor A (α1A), α1-adrenergic receptor B (α1B), α1-adrenergic receptor D (α1D) and G protein-coupled receptor 68 (GPR68) protein levels in mesenteric and hindlimb arteries of Pkd2 fl/fl and Pkd2 fl/fl :myh11-cre/ERT2 mice. ( B ) Mean data from mesenteric and hindlimb arteries (n = 4 per group).

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Mouse Assay, Western Blot

    Lower blood pressure is sustained in Pkd 2 smKO mice. ( A ) Mean arterial blood pressure (MAP) in Pkd 2 smKO and Pkd2 fl/fl mice (n = 6 per group). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Lower blood pressure is sustained in Pkd 2 smKO mice. ( A ) Mean arterial blood pressure (MAP) in Pkd 2 smKO and Pkd2 fl/fl mice (n = 6 per group). * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Mouse Assay

    Arterial myocyte PKD2 knockout attenuates vasoconstriction and arterial wall remodeling during hypertension. ( A ) Mean phenylephrine-induced vasoconstriction in pressurized (80 mmHg) mesenteric arteries from angiotensin II-treated mice ( Pkd2 fl/fl , n = 7–8; Pkd 2 smKO, n = 8).

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Arterial myocyte PKD2 knockout attenuates vasoconstriction and arterial wall remodeling during hypertension. ( A ) Mean phenylephrine-induced vasoconstriction in pressurized (80 mmHg) mesenteric arteries from angiotensin II-treated mice ( Pkd2 fl/fl , n = 7–8; Pkd 2 smKO, n = 8).

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Knock-Out, Mouse Assay

    Myocyte PKD2 knockout does not alter phenylephrine or angiotensin II-induced vasoconstriction in hindlimb arteries. ( A ) Mean passive diameter at 80 mmHg of first-order gastrocnemius arteries (G) and third-, fourth-and fifth-order mesenteric arteries (M) ( Pkd2 fl/fl : G, n = 5; M3 rd n = 4; M4 th n = 5; M5 th n = 5 and Pkd 2 smKO: G, n = 5; M3rd n = 7; M4th n = 4; M5th n = 5). ( B ) Mean data for 60 mM K+-induced constriction in pressurized (100 mmHg) gastrocnemius arteries from Pkd2 fl/fl (n = 4) and Pkd 2 smKO (n = 4) mice. * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Myocyte PKD2 knockout does not alter phenylephrine or angiotensin II-induced vasoconstriction in hindlimb arteries. ( A ) Mean passive diameter at 80 mmHg of first-order gastrocnemius arteries (G) and third-, fourth-and fifth-order mesenteric arteries (M) ( Pkd2 fl/fl : G, n = 5; M3 rd n = 4; M4 th n = 5; M5 th n = 5 and Pkd 2 smKO: G, n = 5; M3rd n = 7; M4th n = 4; M5th n = 5). ( B ) Mean data for 60 mM K+-induced constriction in pressurized (100 mmHg) gastrocnemius arteries from Pkd2 fl/fl (n = 4) and Pkd 2 smKO (n = 4) mice. * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Knock-Out, Mouse Assay

    PKD2 knockout does not alter phenylephrine (PE)-activated ICat in isolated hindlimb artery myocytes. ( A ) Representative I-V relationships recorded between −100 and +100 mV in the same hindlimb artery myocytes of Pkd2 fl/fl or Pkd 2 smKO mice in control and PE (10 µM). ( B ) Mean data for current density at −100 and +100 mV ( Pkd2 fl/fl , n = 6 and Pkd 2 smKO, n = 6). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: PKD2 knockout does not alter phenylephrine (PE)-activated ICat in isolated hindlimb artery myocytes. ( A ) Representative I-V relationships recorded between −100 and +100 mV in the same hindlimb artery myocytes of Pkd2 fl/fl or Pkd 2 smKO mice in control and PE (10 µM). ( B ) Mean data for current density at −100 and +100 mV ( Pkd2 fl/fl , n = 6 and Pkd 2 smKO, n = 6). * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Knock-Out, Isolation, Mouse Assay

    Genotyping of mouse lines. Ethidium bromide gel illustrating PCR products in vasculature of C57BL/6J (WT) mice and tamoxifen-treated Pkd2 fl/fl and Pkd2 fl/fl :myh11cre/ERT2 mice.

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: Genotyping of mouse lines. Ethidium bromide gel illustrating PCR products in vasculature of C57BL/6J (WT) mice and tamoxifen-treated Pkd2 fl/fl and Pkd2 fl/fl :myh11cre/ERT2 mice.

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Polymerase Chain Reaction, Mouse Assay

    PKD2 protein is lower in aorta and mesenteric and hindlimb arteries from tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( A ) Western blots illustrating PKD2 protein was lower in mesenteric arteries of tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice, whereas other proteins were similar. ( B ) Mean data for proteins in hindlimb arteries of Pkd2 smKO mice (n = 4–6). ( C ) Western blots of proteins in aorta. Cav1.2L, full-length Cav1.2; Cav1.2S, short Cav1.2. ( D ) Mean data from aorta (n = 4). * indicates p

    Journal: eLife

    Article Title: Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure

    doi: 10.7554/eLife.42628

    Figure Lengend Snippet: PKD2 protein is lower in aorta and mesenteric and hindlimb arteries from tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice. ( A ) Western blots illustrating PKD2 protein was lower in mesenteric arteries of tamoxifen-treated Pkd2 fl/fl :myh11-cre/ERT2 mice, whereas other proteins were similar. ( B ) Mean data for proteins in hindlimb arteries of Pkd2 smKO mice (n = 4–6). ( C ) Western blots of proteins in aorta. Cav1.2L, full-length Cav1.2; Cav1.2S, short Cav1.2. ( D ) Mean data from aorta (n = 4). * indicates p

    Article Snippet: After blocking with 5% BSA, cells were incubated with mouse monoclonal anti-PKD2 antibody (Santa Cruz) overnight at 4°C.

    Techniques: Mouse Assay, Western Blot

    Protein kinase D (PKD)–2 expression in human gliomas. (A–C) A set of 53 human gliomas was tested for PKD2 expression by immunohistochemistry. Staining intensity of glioma cells was defined as low (1), intermediate (2), and high (3) using

    Journal: Neuro-Oncology

    Article Title: Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation

    doi: 10.1093/neuonc/nor084

    Figure Lengend Snippet: Protein kinase D (PKD)–2 expression in human gliomas. (A–C) A set of 53 human gliomas was tested for PKD2 expression by immunohistochemistry. Staining intensity of glioma cells was defined as low (1), intermediate (2), and high (3) using

    Article Snippet: Whole-cell extracts (2000 μg) were incubated with 2 µg of PKD2 antibody (Bethyl Laboratories) at 4°C for 1 h, and then 80 μL of protein A sepharose beads was added and incubated for 1 h. Immobilized proteins were washed extensively, resuspended in Laemmli buffer, and subjected to SDS-PAGE and Western blotting, as described above.

    Techniques: Expressing, Immunohistochemistry, Staining

    siRNA-mediated depletion of protein kinase D (PKD)–2 inhibits glioblastoma tumor formation in vivo. (A) U87 glioblastoma cells (1 × 10 6 ) transfected with scrambled siRNA or specific PKD2-siRNA oligonucleotides (siPKD2) were implanted inside

    Journal: Neuro-Oncology

    Article Title: Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation

    doi: 10.1093/neuonc/nor084

    Figure Lengend Snippet: siRNA-mediated depletion of protein kinase D (PKD)–2 inhibits glioblastoma tumor formation in vivo. (A) U87 glioblastoma cells (1 × 10 6 ) transfected with scrambled siRNA or specific PKD2-siRNA oligonucleotides (siPKD2) were implanted inside

    Article Snippet: Whole-cell extracts (2000 μg) were incubated with 2 µg of PKD2 antibody (Bethyl Laboratories) at 4°C for 1 h, and then 80 μL of protein A sepharose beads was added and incubated for 1 h. Immobilized proteins were washed extensively, resuspended in Laemmli buffer, and subjected to SDS-PAGE and Western blotting, as described above.

    Techniques: In Vivo, Transfection

    Depletion of protein kinase D (PKD)–2 inhibits glioblastoma cell proliferation in vitro. (A) Glioblastoma cells (1 × 10 4 ) seeded in 6-well plates were transfected on 2 consecutive days with PKD2-siRNA oligo B (siPKD2) or a scrambled oligonucleotide.

    Journal: Neuro-Oncology

    Article Title: Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation

    doi: 10.1093/neuonc/nor084

    Figure Lengend Snippet: Depletion of protein kinase D (PKD)–2 inhibits glioblastoma cell proliferation in vitro. (A) Glioblastoma cells (1 × 10 4 ) seeded in 6-well plates were transfected on 2 consecutive days with PKD2-siRNA oligo B (siPKD2) or a scrambled oligonucleotide.

    Article Snippet: Whole-cell extracts (2000 μg) were incubated with 2 µg of PKD2 antibody (Bethyl Laboratories) at 4°C for 1 h, and then 80 μL of protein A sepharose beads was added and incubated for 1 h. Immobilized proteins were washed extensively, resuspended in Laemmli buffer, and subjected to SDS-PAGE and Western blotting, as described above.

    Techniques: In Vitro, Transfection

    Protein kinase D (PKD)–2 expression in a normal human autopsy brain of a 74-year-old man. (A) PKD2 expression in the cortical neuropil of all layers (I-VI) of the occipital neocortex (OCC). (B) High-power magnification of layer III of the occipital

    Journal: Neuro-Oncology

    Article Title: Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation

    doi: 10.1093/neuonc/nor084

    Figure Lengend Snippet: Protein kinase D (PKD)–2 expression in a normal human autopsy brain of a 74-year-old man. (A) PKD2 expression in the cortical neuropil of all layers (I-VI) of the occipital neocortex (OCC). (B) High-power magnification of layer III of the occipital

    Article Snippet: Whole-cell extracts (2000 μg) were incubated with 2 µg of PKD2 antibody (Bethyl Laboratories) at 4°C for 1 h, and then 80 μL of protein A sepharose beads was added and incubated for 1 h. Immobilized proteins were washed extensively, resuspended in Laemmli buffer, and subjected to SDS-PAGE and Western blotting, as described above.

    Techniques: Expressing