Journal: American Journal of Physiology - Cell Physiology

Article Title: Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain

doi: 10.1152/ajpcell.00305.2017

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: Snap23 111 202 Paraffin–TEG 1:500 Rabbit SYnaptic SYstems Syntaxin-3 sc-47437 Paraffin–TEG 1:100 Goat Santa Cruz Biotech.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain

doi: 10.1152/ajpcell.00305.2017

Figure Lengend Snippet: Exocyst components and SNAREs identified by MS analysis of FACS-isolated CPECs

Article Snippet: Snap23 111 202 Paraffin–TEG 1:500 Rabbit SYnaptic SYstems Syntaxin-3 sc-47437 Paraffin–TEG 1:100 Goat Santa Cruz Biotech.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain

doi: 10.1152/ajpcell.00305.2017

Figure Lengend Snippet: Localization of SNARE proteins in CPECs. A: immunofluorescence labeling of mouse brain sections for the luminal target SNARE syntaxin-3. The left panel is a lower-magnification overview, while the right panel is a higher-magnification micrograph. Syntaxin-3 immunolabeling was confined to the luminal membrane domain. B: immunofluorescence labeling for the basolateral target SNARE syntaxin-4 (Abcam). The left panel shows the labeling pattern in a mouse brain section, whereas the right panel represents immunolabeling of free-floating CP tissue. Syntaxin-4 immunolabeling was mainly confined to the luminal membrane domain. C: immunofluorescence labeling using a different anti-syntaxin-4 antibody (SySy). The left panel is a lower-magnification overview, while the right panel exemplifies a higher-magnification micrograph. The immunoreactivity was most pronounced corresponding to the luminal plasma membrane domain, although the expected basolateral staining was also observed. D: immunolabeling of mouse brain sections for the vesicle SNARE Snap23. The left panel is a lower-magnification overview, while the right panel is a higher-magnification micrograph. Snap23 immunoreactivity was confined to the luminal plasma membrane domain. Except for the image from free-floating tissue staining, the fluorescence micrographs are overlaid on the corresponding DIC images. Nuclei are visualized by Topro-3 (blue). Arrows indicate the luminal plasma membrane, whereas arrowheads mark the basal plasma membrane. The images are representative for micrographs from at least 3 mice.

Article Snippet: Snap23 111 202 Paraffin–TEG 1:500 Rabbit SYnaptic SYstems Syntaxin-3 sc-47437 Paraffin–TEG 1:100 Goat Santa Cruz Biotech.

Techniques: Immunofluorescence, Labeling, Immunolabeling, Membrane, Clinical Proteomics, Staining, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain

doi: 10.1152/ajpcell.00305.2017

Figure Lengend Snippet: Schematic representation of the localization of basic luminal and basolateral membrane domain markers in CPECs. The cell-cell adhesive cadherins, P-cadherin, and N- and/or N2-cadherin (P-cad and N-cad, respectively) localize to the lateral membrane domains including the basal labyrinths. The Par complex (including Par-3, PKCζ, Cdc42, and Par-6) and the Crumbs complex (including Crb3, Patj, and Pals1) are both localized in the luminal membrane domain and/or tight junction area. Par-1 is like the Scribble complex (Scrib, Lgl1, and Dlg1) localized towards the basolateral pole of the cells, whereas Lgl2 is mainly observed in the luminal membrane domain. The polarized plasma membrane phospholipids PIP2 and PIP3 are expressed in the luminal and basolateral membrane domains, respectively, and the phosphatase involved in converting PIP3 to PIP2, Pten, is localized luminally as PIP2. The plasma membrane target SNAREs syntaxin-3 (Stx3) and syntaxin-4 (Stx4) are both mainly localized to the luminal membrane domain, along with the vesicle SNARE Snap23. The luminal expression of Lgl2 and syntaxin-4 with the interacting Snap23 is an unanticipated result.

Article Snippet: Snap23 111 202 Paraffin–TEG 1:500 Rabbit SYnaptic SYstems Syntaxin-3 sc-47437 Paraffin–TEG 1:100 Goat Santa Cruz Biotech.

Techniques: Membrane, Adhesive, Clinical Proteomics, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: (A) Confocal microscopy images of human placenta expressing endogenous CPPED1, PAK4, PIK3R2 and AKT1. Colocalization fluorescence image of CPPED1 (green) and PAK4 (red) (top row), CPPED1 (green) and (PIK3R2) (red) (middle row), and CPPED1 (green) and AKT1 (red) (bottom row). Middle panel shows green channel, red channel and bright field image separately of the left panel. Panels on the right show negative controls treated the same way as samples but with primary antibody omitted. Objective used was HC PL APO 63×/1.40 OIL CS2 DIC (oil). Scale bar represents 10 µm. (B) Zoom in view of the images shown in A (left panels). (C) Confocal microscopy images of transfected HEK293T cells coexpressing CPPED1 with PIK3R2 and PAK4. Fluorescence image of HEK293T cells coexpressing CPPED1‐VN and PIK3R2‐VC (top row, right panel) and CPPED1‐VN and PAK4‐VC (bottom row, right panel). VN is the N‐terminal fragment of Venus yellow fluorescence protein (YFP), and VC is the C‐terminal fragment of Venus YFP. CPPED1 or JUN (control) was fused to the N‐terminal fragment of Venus, while PIK3R2, PAK4 or FOS (control) was fused to the C‐terminal fragment of Venus. If the two coexpressing proteins interact, N‐ and C‐terminal fragments of Venus YFP are brought together, which results in stable intact Venus YFP and a fluorescence signal. As controls, CPPED1‐VN and FOS‐VC (top row, left panel) and JUN‐VN and PIK3R2‐VC (top row, middle panel) were coexpressed in HEK293T cells. Similarly, JUN‐VN and PAK4‐VC (bottom row, middle panel) were coexpressed in HEK293T cells. Objective: UPLSAPO 60×/1.35 (oil). Scale bar same for all images

Article Snippet: In the reaction mixture (50 μl), the final concentrations of CPPED1 (purified by us), PAK4 (TP302302; Origene) and PIK3R2 (009‐001‐S95S; Rockland) were 0.13 μM, 0.16 μM and 0.15 μM, respectively.

Techniques: Confocal Microscopy, Expressing, Fluorescence, Transfection, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: CPPED1‐interacting proteins obtained by human proteome microarray. Above‐threshold interactions are listed in order of highest to lowest binding affinity with CPPED1

Article Snippet: In the reaction mixture (50 μl), the final concentrations of CPPED1 (purified by us), PAK4 (TP302302; Origene) and PIK3R2 (009‐001‐S95S; Rockland) were 0.13 μM, 0.16 μM and 0.15 μM, respectively.

Techniques: Microarray, Binding Assay, Derivative Assay, RNA Binding Assay, Ubiquitin Proteomics, Transduction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: KEGG pathway analysis of CPPED1 binding partners identified by human proteome microarray. Significant terms ( P <.05) are shown

Article Snippet: In the reaction mixture (50 μl), the final concentrations of CPPED1 (purified by us), PAK4 (TP302302; Origene) and PIK3R2 (009‐001‐S95S; Rockland) were 0.13 μM, 0.16 μM and 0.15 μM, respectively.

Techniques: Binding Assay, Microarray

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: Coimmunoprecipitation of CPPED1, PAK4 and PIK3R2. Myc‐tagged PAK4 and flag‐tagged CPPED1 (left panel) and myc‐tagged PIK3R2 and flag‐tagged CPPED1 (right panel) were transiently expressed in HEK293T cells. Tagged proteins PAK4 and PIK3R2 were coimmunoprecipitated with anti‐myc Sepharose. Except for the negative control, in which anti‐myc Sepharose was replaced by Sepharose, all samples were treated in the same manner. Immunocomplexes were analysed by Western blot (WB), and by using different antibodies for the same immunoprecipitation (IP) sample. Antibodies used were anti‐myc, anti‐flag anti‐CPPED1, anti‐PAK4 and anti‐PIK3R2. The following IP samples are the same: 1 and 3, 2 and 4, 5 and 7, and 6 and 8. For example, in the Western blot, anti‐myc and anti‐flag antibodies were used with sample 1, and anti‐PAK4 and anti‐CPPED1 antibodies were used with sample 3. The original blots are presented in Figs. S8 and S9

Article Snippet: In the reaction mixture (50 μl), the final concentrations of CPPED1 (purified by us), PAK4 (TP302302; Origene) and PIK3R2 (009‐001‐S95S; Rockland) were 0.13 μM, 0.16 μM and 0.15 μM, respectively.

Techniques: Negative Control, Western Blot, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: CPPED1 involvement with PI3K‐AKT pathway. Schematic view of activated (A) and inactivated (B) PI3K‐AKT pathway. PI3K‐AKT is a conserved pathway that is involved in many types of cellular processes. When growth factor (GF) binds to receptor tyrosine kinase (RTK), RTK tyrosine residues are phosphorylated. Regulatory subunit p85 binds to phosphorylated tyrosine and brings catalytic subunit p110 adjacent to substrate PIP2. Once PIP3 is formed from PIP2 by PI3K, pleckstrin homology (PH) domains of PDK1, PDK2 and AKT1 bind to PIP3, which results in phosphorylation and activation of AKT1. PDK1 phosphorylates Thr‐308. Ser‐473 is phosphorylated by various proteins such as PDK2, integrin‐linked kinase (ILK), mechanistic target of rapamycin complex (mTORC2) and DNA‐dependent protein kinase (DNA‐PK). , PAK4 has been shown to bind to p85α (also known as PIK3R1) and is required for maximal phosphorylation of AKT1. By contrast, protein phosphatase 2A (PP2A), protein phosphatase 1 (PP1), PH domain leucine‐rich repeat protein phosphatase 2 (PHLPP2) and CPPED1 are known to dephosphorylate Ser‐473 and inactivate AKT1. Among other functions, activated AKT1 phosphorylates transcription factor FOXO1, which results in nuclear exclusion of phosphorylated FOXO1. This might also prevent formation of the FOXO‐progesterone receptor (PR) transcription factor complex. , , By contrast, PI3K‐AKT pathway inhibition allows for formation of the FOXO‐PR complex. We found that CPPED1 bound p85β (also known as PIK3R2) and PAK4, which could have an effect on the PI3K‐AKT Pathway. We also found that CPPED1 mediated the effect of progesterone on the expression of a set of genes.

Article Snippet: In the reaction mixture (50 μl), the final concentrations of CPPED1 (purified by us), PAK4 (TP302302; Origene) and PIK3R2 (009‐001‐S95S; Rockland) were 0.13 μM, 0.16 μM and 0.15 μM, respectively.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Expressing

Journal: bioRxiv

Article Title: IR-B deficiency and fatty acid dysregulation accelerate prostate cancer progression via PI3K/AKT signaling

doi: 10.64898/2026.01.30.702723

Figure Lengend Snippet: (A) Volcano plot showing differentially expressed genes between LL H (control) and pLL +H prostate DLP lobes. (B) Hierarchical clustering and heatmap of significantly differentially expressed genes; enriched gene ontology (GO) terms indicated. (C) Bubble plot of enriched KEGG pathways for upregulated genes. (D) Real-time PCR quantification of IRS-1, IRS-2, IRS-3, and IGF2 mRNA in LL H and pLL +H prostate DLP lobes. mean ± SEM. (E) Western blot analysis of p- IR, total IR, p- IRS, total IRS1, p- AKT, total AKT, and PI3K protein in LL, pLL + , LL H and pLL +H prostate DLP lobes. (F) Heatmap showing differential metabolites between pLL +H and LL H mice. (G) Classification of metabolite type in prostate DLP lobes of LL H and pLL +H mice. (H) Volcano plot of significantly differential metabolites; fatty acids highlighted.

Article Snippet: The following primary antibodies were used: IRS, phospho-IRS1(Tyr896), GSK3β and phospho-GSK3β(Ser9), GPR120, GLUT1, Bcl-2, BAX (Abbkine, USA); IR and phospho-IR, AR, AKT1 and phospho-AKT, PI3K p85 alpha, GAPDH (Proteintech, Wuhan, China); Ki-67 (Cell Signaling Technology, USA); GLUT12 (Boster Bioengineering Co., Ltd., Wuhan); GSK3α/β and phospho-GSK3β (Y216) and phosphor-GSK3α (Y279) (Abcam,USA).

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

Journal: bioRxiv

Article Title: IR-B deficiency and fatty acid dysregulation accelerate prostate cancer progression via PI3K/AKT signaling

doi: 10.64898/2026.01.30.702723

Figure Lengend Snippet: (A & B) Western blot and quantification showing AR expression reduced by ω-3 and increased by ω-6. (C & D) Western blot and quantification of p -PI3K, PI3K, p -AKT, and AKT at 3 and 6 months; ω-3 decreases phosphorylation, ω-6 increases it. (E, F, G, & H) Western blot and quantification of BCL2 and BAX at 3 and 6 months. ω-3 increases pro-apoptotic BAX and decreases anti-apoptotic BCL2, ω-6 shows opposite pattern. (I & J) Western blot of cultured cells treated with EPA, DHA, or AA, corfirming protein expression treads. Data mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following primary antibodies were used: IRS, phospho-IRS1(Tyr896), GSK3β and phospho-GSK3β(Ser9), GPR120, GLUT1, Bcl-2, BAX (Abbkine, USA); IR and phospho-IR, AR, AKT1 and phospho-AKT, PI3K p85 alpha, GAPDH (Proteintech, Wuhan, China); Ki-67 (Cell Signaling Technology, USA); GLUT12 (Boster Bioengineering Co., Ltd., Wuhan); GSK3α/β and phospho-GSK3β (Y216) and phosphor-GSK3α (Y279) (Abcam,USA).

Techniques: Western Blot, Expressing, Phospho-proteomics, Cell Culture

Journal: bioRxiv

Article Title: IR-B deficiency and fatty acid dysregulation accelerate prostate cancer progression via PI3K/AKT signaling

doi: 10.64898/2026.01.30.702723

Figure Lengend Snippet: Left: Dysregulation of IR isoforms mediates cell proliferation and glucose metabolism via PI3K/AKT signaling in Hi-Myc mice. Right: IR-B knockout leads to glucose metabolism disorder and compensatory increase of ω-3 polyunsaturated fatty acids, which upregulate GPR120 expression and enhance glucose uptake through GLUT1.

Article Snippet: The following primary antibodies were used: IRS, phospho-IRS1(Tyr896), GSK3β and phospho-GSK3β(Ser9), GPR120, GLUT1, Bcl-2, BAX (Abbkine, USA); IR and phospho-IR, AR, AKT1 and phospho-AKT, PI3K p85 alpha, GAPDH (Proteintech, Wuhan, China); Ki-67 (Cell Signaling Technology, USA); GLUT12 (Boster Bioengineering Co., Ltd., Wuhan); GSK3α/β and phospho-GSK3β (Y216) and phosphor-GSK3α (Y279) (Abcam,USA).

Techniques: Knock-Out, Expressing