Journal: Frontiers in Pharmacology

Article Title: Jianpi Yangzheng Xiaozheng decoction alleviates gastric cancer progression via suppressing exosomal PD-L1

doi: 10.3389/fphar.2023.1159829

Figure Lengend Snippet: JPYZXZ reduced the number of MDSCs in tumor bearing mice. (A,B) Percentage of MDSCs (CD45 + CD11b+Gr1+) in blood determined using flow cytometry. (C,D) Percentage of MDSCs (CD45 + CD11b+Gr1+) in tumors determined using flow cytometry. (E) Immunofluorescence staining for DAPI (nucleus), CD11b, Gr-1, and PD-L1 in tumor tissue sections from the xenograft tumor model. Data are represented as the mean ± SEM, n = 5 mice or n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The single-cell suspensions (1 × 10 7 cells/mL) were stained with fluorochrome-coupled antibodies AF488-CD45 (Elabscience, E-AB-F1136L), PE/Dazzle™594-CD11b (BioLegend, 101256), and APC-Gr-1 (BioLegend, 108412) for 30 min at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 1: DLC3 depletion enhances TGF-β-induced matrix degradation in a RhoB-dependent manner.

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques:

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 2: DLC3 functions as a GAP protein for endosomal RhoB. (A-D) MCF10A cells were pretreated

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques:

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 3: SNX27 recruits DLC3 to endomembranes. (A) HEK293T cells were transiently transfected with

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques: Transfection

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 4: DLC3 depletion traps MT1-MMP in early endosomes. MDA-MB-231 cells stably

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques: Stable Transfection

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 5: DLC3 knockdown enhances MT1-MMP surface levels and exocytosis. MDA-MB-231 cells

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques: Knockdown

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 6: DLC3 depletion enhances matrix degradation via Rab4-dependent recycling of MT1-MMP.

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques:

Journal: Journal of cell science

Article Title: DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

doi: 10.1242/jcs.223172

Figure Lengend Snippet: Figure 7: Schematic representation of the proposed role of DLC3 in the regulation of endosomal RhoB

Article Snippet: Actin (AC-40) Mouse mAb Sigma-Aldrich A4700 1:500 (WB) c-myc (9E10) Mouse mAb Dr. Böttinger, IZI, Uni Stuttgart 1:1000 (WB) Cortactin (4F11) Mouse mAb Millipore 05-180 1:200 (IF) DLC3 (E-2) Mouse mAb Santa Cruz sc-166725 1:500 (WB) E-Cadherin Rabbit mAb Cell Signaling #3195 1:1000 (WB) EEA1 Rabbit pAb Cell Signaling #2411 1:100 (IF) Flag (M2) Mouse mAb Sigma-Aldrich F1804 1:1000 (WB) GAPDH Rabbit pAb Sigma-Aldrich G9545 1:5000 (WB) mCherry Rabbit pAb Abcam ab167453 1:1000 (WB) N-Cadherin Mouse mAb BD 610921 1:250 (IF), 1:1000 (WB) Rab7 (D95F2) XP Rabbit mAb Cell Signaling #9367 1:100 (IF) RhoA (26C4) Mouse mAb Santa Cruz sc-418 1:500 (WB) RhoB Rabbit pAb Cell Signaling #2098 1:500 (WB) SNX27 (1C6) Mouse mAb Abcam ab77799 1:200 (IF), 1:500 (WB) Vimentin Mouse mAb BD 550513 1:5000 (WB) α-tubulin (B-5-1-2) Mouse mAb Sigma-Aldrich T5168 1:5000 (WB) giantin Rabbit pAB Abcam ab24586 1:1000 (IF) Table S2: Conjugated antibodies and dilutions

Techniques:

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 4. A, expression of the ST6Gal I in the T200 cell line, which does not express CD45, did not result in increased SNA binding. Nine clones expressing ST6Gal I mRNA were examined, but none demon- strated increased SNA binding by flow cytometry; clone SNA.T1 is shown for example. C.T1 is one of nine control clones transfected with vector alone. B, SNA blotting of whole cell extracts of C.T1 and SNA.T1 cells did not demonstrate any differences in staining between the two clones. C, T200 clones express ST6Gal I mRNA and protein. RT-PCR and immunoblot analysis of nine clones demonstrated ST6Gal I expres- sion, as shown for the SNA.T1 clone, with no ST6Gal I expression in any of the controls, as shown for the C.T1 clone. The samples are represent- ative of all 18 clones examined. The expressed protein is enzymatically active, as demonstrated by the addition of sialic acid to asialofetuin. Asialofetuin was incubated with lysates of C.T1 or SNA.T1 cells and precipitated with anti-fetuin, and 2,6-linked sialic acid was detected by SNA blotting. Weak SNA reactivity of fetuin incubated with extract of C.T1 cells may reflect the addition of 2,6-linked sialic acid to O-glycans, because no SNA reactivity was detected with the asialofe- tuin acceptor substrate alone (not shown). Densitometric analysis of the SNA-binding bands was performed; the ratio of SNA binding to fetuin incubated with SNA.T1 extract compared with C.T1 extract was 6.3:1.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Clone Assay, Flow Cytometry, Control, Transfection, Plasmid Preparation, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 3. The ST6Gal I preferentially sialylates N-glycans on CD45. A, total SNA-binding glycoproteins were precipitated from con- trol clones transfected with vector alone (lanes C.2 and C.4) or from the SNA.1 clone expressing the ST6Gal I. Precipitated glycoproteins were probed with biotinylated SNA. The only significant difference in the profile of SNA binding glycoproteins was an increase in a band with a mass of 200 kDa (arrow). B, the SNA reactive band is CD45. The cells were cultured in 2 mM DMNJ, as above, or in medium alone. The cell lysates were precipitated with CD45 antibody or SNA (indicated below) and probed with CD45 antibody. The band with increased SNA staining reacts with both SNA and antibody to CD45. In addition, the increased SNA binding to CD45 is abolished by pretreatment with DMNJ, which blocks synthesis of complex N-glycans. In both blots, the width of the CD45 band is diminished in DMNJ-treated cells compared with cells expressing the ST6Gal I, as a result of decreased complexity of glyco- sylation. C, increased SNA binding to CD45 results from sialylation of N-glycans. CD45 was detected in whole cell lysates of SNA.9 cells by immunoblotting (top panel). The CD45 bands were excised and incu- bated with or without PNGase F, as indicated, and reprobed with SNA-biotin. Removal of N-glycans from CD45 by PNGase F treatment reduced SNA binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Clone Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 5. ST6Gal I expression inhibits galectin-1-induced segre- gation of CD45. C.4 or SNA.9 cells were treated with galectin-1 or buffer control and fixed, and cell surface CD45 was detected by immu- nofluorescence. A, cells were analyzed by confocal microscopy to detect CD45 segregation. B, the percentage of cells demonstrating segregation of CD45 was scored by counting 50 cells in six fields. The cells were treated with buffer control (open bars) or galectin-1 (shaded bars). The SNA.9 cells demonstrated a marked reduction in CD45 segregation, compared with CD45 segregation in control C.4 cells. The percentage of cell death for a parallel sample is indicated by the numbers above each bar.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Control, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: The ST6Gal I Sialyltransferase Selectively ModifiesN-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death

doi: 10.1074/jbc.m209595200

Figure Lengend Snippet: FIG. 6. ST6Gal I expression inhibits galectin-1-induced modu- lation of CD45 protein-tyrosine phosphatase activity. A, CD45 is the major PTP in PhaR2.1 cells. Whole cell lysates of the CD45 paren- tal cell line, PhaR2.1, and the CD45 derivative T200, were assayed for PTP activity in the presence (solid bar) or absence (open bar) of the PTP inhibitor bp V (phen). PTP activity was measured by the release of p-nitrophenol, detected at 415 nm. B, ST6Gal I expression abrogates the decrease in PTP activity triggered by binding of galectin-1. C.2 cells (open symbols) and SNA.9 cells (closed symbols) were incubated with 30 g of galectin-1 for the indicated times at 37 °C. At the indicated times, the cells were lysed, and PTP activity in whole cell lysates was meas- ured as described under “Experimental Procedures,” in the presence (squares) or absence (circles) of the PTP inhibitor bpV (phen). C.2 cells demonstrate a 40% reduction in PTP activity 1 min after galectin-1 binding. SNA.9 cells demonstrate no change in PTP activity after ga- lectin-1 binding.

Article Snippet: PNGase F Digestion of CD45—Cell lysates (106 cells) were separated by SDS-PAGE, blotted to nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation

Journal: Frontiers in Immunology

Article Title: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

doi: 10.3389/fimmu.2018.00815

Figure Lengend Snippet: PKCζ is an NSM downstream effector. (A,B) Jurkat-ΔNSM and control cells were stimulated with PMA/ionomycin. Accumulation of pPKCδ, pPKCθ, and pPKCζ/λ was determined over time (A) and IL-2-release after 48 h (B) . (C) Accumulation of pPKCζ/λ was determined in Jurkat-ΔNSM and control cells stimulated with α-CD3 over time. (D) Detergent resistant membrane (DRM) domain association of PKCζ and PKCθ was determined in unstimulated (upper panels) and in 5 min α-CD3-stimulated (bottom panels) parental and Jurkat-ΔNSM cells (detection of Lck was used to identify DRM fractions). (E) PKCζ and F-actin were co-detected in CTRL (IF panels, upper row) or NSM KD cells (IF panels, bottom row) 15 min after stimulation with α-CD3-coated beads. The percentage of cells polarizing PKCζ (left graph) and relative polarization [immune synapse (IS)/cytoplasm] in individual cells were quantified (right graph). PKCζ IS localization was visualized by en face view (representative examples are shown in IF pictures on the right side). Size bars: 5 µM.

Article Snippet: PKCζ [rabbit mAb, clone EP1490(2)] from Abcam; CD3ε (rat mAb, clone CD3–12), LAT (rabbit polyclonal, FL-233), and Lck (mouse mAb, clone 73A5) antibodies from Santa Cruz were used to detect DRM associated PKCζ.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

doi: 10.3389/fimmu.2018.00815

Figure Lengend Snippet: Ceramide supplementation rescues PKCζ detergent resistant membrane (DRM) association and microtubule-organizing center (MTOC) polarization in α-CD3-stimulated NSM-depleted T cells. (A) Parental and Jurkat-ΔNSM cells were loaded with functionalized ω-C16-ceramide, DIBO488-clicked and analyzed by IF [ (A) insets] and subjected to floatation gradient centrifugation after which fractions containing DRM resident proteins Lck, LAT, CD3ζ, and CD3ε (serving as DRM markers) were identified by Western blot (upper panels) and accumulation of DIBO488 was measured in all fractions by fluorescence reader (bottom panels). (B) DRM domain association of PKCζ was determined after 5 min of α-CD3-stimulation of parental and Jurkat-ΔNSM cells pre-loaded with ω-C 16 -ceramide (detection of Lck was used to identify DRM fractions). (C) Jurkat-ΔNSM loaded with ω-C 16 -cermide or pretreated with the GSK3-inhibitor indirubin-3′-monoxime were α-CD3 stimulated. Graph shows the percentage of MTOC polarizing cells quantified after 15 min of stimulation as described in Figures B,C. Representative IF pictures for each condition are shown. Size bar: 10 µM. (A–C) One representative experiment out of three is shown.

Article Snippet: PKCζ [rabbit mAb, clone EP1490(2)] from Abcam; CD3ε (rat mAb, clone CD3–12), LAT (rabbit polyclonal, FL-233), and Lck (mouse mAb, clone 73A5) antibodies from Santa Cruz were used to detect DRM associated PKCζ.

Techniques: Gradient Centrifugation, Western Blot, Fluorescence

Journal: Journal of Virology

Article Title: Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

doi: 10.1128/jvi.00189-10

Figure Lengend Snippet: FIG. 2. Specific interaction of UL112-113 p84 with UL44 among the six replication core proteins. (A) X-Gal filter assays of yeast cells expressing both the GAL4-DB/UL112-113 (p34, p43, p50, and p84) fusion proteins and the GAL4-A/replication core (UL44, UL54, UL57, UL70, UL102, and UL105) fusion proteins. The cells expressing the GAL4-A proteins alone were used as a control. Green indicates a positive interaction. (B) 293T cells were cotransfected with plasmids encoding HA-tagged UL112-113 proteins and Myc-tagged replication core proteins (UL44, UL54, UL57, UL70, UL102, or UL105). (Top) At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting with anti-HA Ab. (Middle and bottom) Total cell lysates were also immunoblotted with anti-Myc or anti-HA Abs. (C) HF cells were infected with HCMV Towne at an MOI of 2.0. Total cell lysates were prepared 2 days postinfection and immunoprecipitated with M23 Ab specific for the UL112-113 proteins, followed by immunoblotting with anti-PCNA or anti-p84 Abs. Total cell lysates were also immunoblotted with anti-p84 or anti-PCNA Abs to show the protein expression levels.

Article Snippet: Anti-proliferating cell nuclear antigen (PCNA) mouse MAb PC10 was obtained from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Control, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Cell Science

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis

doi: 10.1242/jcs.223453

Figure Lengend Snippet: SOAT1 loss from EMC-deficient cells and attenuated cholesteryl ester formation. (A) Heat map representing the fold change of 117 lipid species in EMC5 (Δ5 #5-4) and EMC6 (Δ6 #3-9) deletion mutants relative to WT as identified by targeted metabolomic analysis ( n ≥4). Lipid species are arranged according to major structural class. The 14 lipid species significantly altered in both Δ5 and Δ6 cells ( P ≤0.05) are indicated (grey circles, black star). NL, neutral lipids; FA, fatty acids; AC, acyl carnitine; NAE, N-acylethanolamines; ST, sterols; PL, phospholipids; LPL, lysophospholipids; SL, sphingolipids; NEL, neutral ether lipids; PLE, phospholipid ethers; LPLE, lysophospholipid ethers. (B) Quantification of free cholesterol and cholesteryl esters in Δ5 and Δ6 cells relative to WT (dashed line). Means±s.e.m. are shown ( n =4). *** P ≤0.001, **** P ≤0.0001 (Student's t -test). (C,D) Western blots of whole-cell lysates (WCL) for Δ5 cells with or without the EMC5 expression vector (C) and Δ6 cells with or without the EMC6 expression vector (D) probed for SOAT1 and indicated EMC subunits. (E) Schematic representation of the dual reporter construct used in F–G. mRNA encoding GFP separated by a P2A sequence from RFP and FLAG-tagged SOAT1. Translation results in ribosome skipping and the generation of GFP and RFP-3xFLAG-SOAT1 at equimolar ratios. Differences in stability between both gene products gives rise to altered RFP:GFP ratios, serving as a sensitive readout for protein stability. (F) Fluorocytometric RFP:GFP ratio in WT and Δ5 cells with or without the EMC5 expression vector and transiently expressing GFP-P2A-RFP-3xFLAG-SOAT1. At 24 h post transfection, cells were treated with MG132 (5 µg/ml) or DMSO (vehicle control) for 8 h and analysed by flow cytometry. EV, empty vector control. (G) Quantification of three independent experiments as performed in F. Means±s.d. are shown ( n =3). **** P ≤0.0001 (Student's t -test). (H) WT and Δ6 cells were exposed to Chol:MBCD (25, 37.5 and 50 µM, 20 h) with or without 10 µM avasimibe (AVA, 20 h) and visualised by staining with Crystal Violet.

Article Snippet: The following antibodies were used in this study: ACC1 (Cell Signaling, Danvers, MA, rabbit pAb, #4190), 1:1000; AlexaFluor R488 anti-mouse IgG (H+L) (Invitrogen, donkey pAb, #A21202), 1:4000 [immunoblotting (IB)], 1:400 [immunofluorescence (IF)]; AlexaFluor R488 anti-rabbit IgG (H+L) (Invitrogen, goat pAb, #A11008), 1:4000 (IB), 1:400 (IF); anti-mouse IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2005), 1:10,000; anti-rabbit IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2030), 1:10,000; EMC1 (rabbit pAb, kind gift of Enilza Espreafico, Sao Paulo, Brazil), 1:1000; EMC2 (Proteintech, Rosemont, IL, rabbit pAb, #25443-1-AP), 1:2000; EMC3 (Abcam, Cambridge, UK, rabbit pAb, #ab175537), 1:500; EMC4 (Abcam, rabbit pAb, #ab123719), 1:1000; EMC5/MMGT1 (Abcam, rabbit pAb, #ab174366), 1:1000; EMC6 (Abcam, rabbit pAb, #ab84902), 1:1000; EMC7 (Abgent, San Diego, CA, rabbit pAb, #AP11145c), 1:500; EMC8 (Abcam, rabbit pAb, #ab180065), 1:500; EMC9 (Abgent, rabbit pAb, #AP5632b), 1:500; EMC10 (Abcam, rabbit pAb, #ab180148), 1:1000; HA (purified from hybridoma, mouse mAb, clone 12CA5), 1:1000; histone H3 (Abcam, rabbit pAb, ab1791), 1:5000; HMGCR (Atlas Antibodies, Bromma, Sweden, mouse mAb, clone CL0260), 1:1000; Hsp70 (Santa Cruz Biotechnology, goat pAb, clone K-20, #sc-1060), 1:1000; LDLR (R&D Systems, Minneapolis, MN, goat pAb, #AF2148), 1:1000; Rap1a (Santa Cruz Biotechnology, rabbit pAb, clone C-17, #sc-1482), 1:1000; SCD1 (Abcam, mouse mAb, clone CD.E10, #ab19862), 1:1000; SM (Proteintech, rabbit pAb, #12544-1-AP), 1:1000; SOAT1 (Santa Cruz Biotechnology, mouse mAb, clone A-11, #sc-136959), 1:1000; SQS (Abcam, rabbit mAb, #ab109723), 1:2000; SQS (Abcam, rabbit mAb, #ab195046), 1:200 (IF), 1:2000 (WB); SREBP2 (R&D Systems, goat pAb, #AF7119), 1:1000; tubulin (Sigma-Aldrich, mouse mAb, #T5168/b-5-1-2), 1:1000; and ubiquitin (Cell Signaling Technologies, rabbit pAb, #3933S), 1:1000.

Techniques: Western Blot, Expressing, Plasmid Preparation, Construct, Sequencing, Transfection, Control, Flow Cytometry, Staining

Journal: Journal of Cell Science

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis

doi: 10.1242/jcs.223453

Figure Lengend Snippet: Model of how EMC-mediated protein biogenesis influences cholesterol homeostasis. (A) The EMC promotes the post-translational insertion of SQS into the ER membrane (i) and maturation of SOAT1 co-translationally (ii). The correct insertion and/or maturation of EMC-dependent proteins maintain cholesterol metabolic pathways by ensuring sufficient expression of enzymes essential for cholesterol biosynthesis and storage in the form of cholesteryl esters (iii, iv). These activities enable the biosynthetic and import pathways to maintain sufficient free cholesterol in cells. Additionally, the EMC is involved directly (or indirectly) in the biogenesis of other putative membrane-bound ‘clients’ with a variety of cellular roles (v). (B) Absence of the EMC precludes SQS insertion, resulting in its premature degradation via the proteasome (vi). Similarly, SOAT1 expression is also reduced co-/post-translationally due to defective biogenesis (vi, vii). The reduction of steady-state levels of both enzymes (depicted in grey) leads to increased utilisation of the mevalonate pathway intermediate FPP for non-sterol isoprenoid biosynthesis (viii), decreased cholesterol biosynthesis and reduced capability to store cholesterol as cholesteryl ester (ix), while at the same time maintaining sufficient free cholesterol to support viability (x). The consequential loss of other EMC clients may be manifest as non-lethal impairments detectable only when targeted or monitored directly.

Article Snippet: The following antibodies were used in this study: ACC1 (Cell Signaling, Danvers, MA, rabbit pAb, #4190), 1:1000; AlexaFluor R488 anti-mouse IgG (H+L) (Invitrogen, donkey pAb, #A21202), 1:4000 [immunoblotting (IB)], 1:400 [immunofluorescence (IF)]; AlexaFluor R488 anti-rabbit IgG (H+L) (Invitrogen, goat pAb, #A11008), 1:4000 (IB), 1:400 (IF); anti-mouse IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2005), 1:10,000; anti-rabbit IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2030), 1:10,000; EMC1 (rabbit pAb, kind gift of Enilza Espreafico, Sao Paulo, Brazil), 1:1000; EMC2 (Proteintech, Rosemont, IL, rabbit pAb, #25443-1-AP), 1:2000; EMC3 (Abcam, Cambridge, UK, rabbit pAb, #ab175537), 1:500; EMC4 (Abcam, rabbit pAb, #ab123719), 1:1000; EMC5/MMGT1 (Abcam, rabbit pAb, #ab174366), 1:1000; EMC6 (Abcam, rabbit pAb, #ab84902), 1:1000; EMC7 (Abgent, San Diego, CA, rabbit pAb, #AP11145c), 1:500; EMC8 (Abcam, rabbit pAb, #ab180065), 1:500; EMC9 (Abgent, rabbit pAb, #AP5632b), 1:500; EMC10 (Abcam, rabbit pAb, #ab180148), 1:1000; HA (purified from hybridoma, mouse mAb, clone 12CA5), 1:1000; histone H3 (Abcam, rabbit pAb, ab1791), 1:5000; HMGCR (Atlas Antibodies, Bromma, Sweden, mouse mAb, clone CL0260), 1:1000; Hsp70 (Santa Cruz Biotechnology, goat pAb, clone K-20, #sc-1060), 1:1000; LDLR (R&D Systems, Minneapolis, MN, goat pAb, #AF2148), 1:1000; Rap1a (Santa Cruz Biotechnology, rabbit pAb, clone C-17, #sc-1482), 1:1000; SCD1 (Abcam, mouse mAb, clone CD.E10, #ab19862), 1:1000; SM (Proteintech, rabbit pAb, #12544-1-AP), 1:1000; SOAT1 (Santa Cruz Biotechnology, mouse mAb, clone A-11, #sc-136959), 1:1000; SQS (Abcam, rabbit mAb, #ab109723), 1:2000; SQS (Abcam, rabbit mAb, #ab195046), 1:200 (IF), 1:2000 (WB); SREBP2 (R&D Systems, goat pAb, #AF7119), 1:1000; tubulin (Sigma-Aldrich, mouse mAb, #T5168/b-5-1-2), 1:1000; and ubiquitin (Cell Signaling Technologies, rabbit pAb, #3933S), 1:1000.

Techniques: Membrane, Expressing