Journal: British Journal of Cancer

Article Title: Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma

doi: 10.1038/bjc.2011.311

Figure Lengend Snippet: The 18 downregurated genes in miR-874 transfectants

Article Snippet: TaqMan probes and primers for PPP1CA (P/N: Hs00267568_m1), PAAF1 (P/N: Hs00228523_m1), TGOLN2 (P/N: Hs00197728_m1) and GUSB (P/N: Hs99999908_m1) internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Binding Assay

Journal: British Journal of Cancer

Article Title: Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma

doi: 10.1038/bjc.2011.311

Figure Lengend Snippet: Expression levels of three candidate genes of miR-874 target were measured by real-time RT–PCR. ( A , upper) PPP1CA mRNA expression levels in MSSCC clinical specimens. ( B , upper) PAAF1 mRNA expression levels in MSSCC clinical specimens. ( C , upper) TGOLN2 mRNA expression levels in MSSCC clinical specimens. Real-time RT–PCR showed that each of the three genes in tumour tissues was expressed at higher levels than that in the normal tissues. GUSB was used as an internal control. ( A , B , and C , lower) Significant inverse correlations between each of the genes and the level of miR-874 expression were shown.

Article Snippet: TaqMan probes and primers for PPP1CA (P/N: Hs00267568_m1), PAAF1 (P/N: Hs00228523_m1), TGOLN2 (P/N: Hs00197728_m1) and GUSB (P/N: Hs99999908_m1) internal control were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α

doi: 10.1038/s41467-021-27597-7

Figure Lengend Snippet: a Western blot analysis of endogenous and ectopic IRE1α variant expression in MDA-MB-231 cells harboring Dox-inducible IRE1α shRNA stably transfected with transgenic WT or R887A mutant versions of IRE1α-GFP. b Immunoblot analysis of MDA-MB-231 cells after treatment with Tg (100 nM, 4 h) followed by DSS crosslinking. Left panel shows parental shIRE1α cl.12 cell line. Right panel shows IRE1α WT and R887A rescues of Doxycycline-treated cl.12 cells with endogenous IRE1α knockdown. c RT-qPCR analysis of IRE1α RNase targets CD59, TGOLN2 (RIDD), and TNFAIP8L1, SNN, and SIX2 (RIDDLE). Ct values for XBP1 in sample shIRE1 cl.1 prior to Tg treatment were >34, precluding ratio calculations and were therefore not plotted. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. A 2-way ANOVA test was used to calculate p-values for CD59 and TGOLN2, and an unpaired t -test for the remaining targets. d Analysis of cell viability by Cell-Titer Glo after Dox treatment for 7 days on Ultra-Low Attachment (ULA) plates. n = 2 biologically independent experiments. Data are presented as mean values ± SEM. e Model depicting IRE1α’s principal modes of endoribonuclease function and their underlying phospho-oligomeric states during ER stress. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: We prepared T7 RNA transcripts from cDNA templates chosen based upon functional relevance coupled with optimal length for the ribonucleolytic reaction (~0.5–2 kb). cDNA constructs encoding XBP1 (#HG10751-UT), DGAT2 (#HG14114-G), CD59 (#HG12474-UT), TGOLN2 (#HG17252-UT), SIX2 (#HG21116-UT), CFAP45 (#HG22377-UT), MFAP2 (#HG16644-UT), PIGQ (#HG22757-UT), BMP4 (#HG10609-UT), BCAM (#HG10238-UT), SNN (#HG23279-U), GBA (#HG12038-UT), WT1 (#HG12282-UT), CCDC69 (#HG27177-U), AIM2(#HG11654-UT) were from Sino Biological, and BLOC1S1 (#RC224412), TNFAIP8L1 (#RC203912) from Origene. cDNA was amplified using T7 forward primers, and subsequently in vitro transcribed using HiScribe™ T7 Quick High Yield RNA Synthesis Kit from NEB (#E2050S).

Techniques: Western Blot, Variant Assay, Expressing, shRNA, Stable Transfection, Transfection, Transgenic Assay, Mutagenesis, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Decoding non-canonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α

doi: 10.1038/s41467-021-27597-7

Figure Lengend Snippet: a Western blot analysis of endogenous and ectopic IRE1α variant expression in MDA-MB-231 cells harboring Dox-inducible IRE1α shRNA stably transfected with transgenic WT or R887A mutant versions of IRE1α-GFP. b Immunoblot analysis of MDA-MB-231 cells after treatment with Tg (100 nM, 4 h) followed by DSS crosslinking. Left panel shows parental shIRE1α cl.12 cell line. Right panel shows IRE1α WT and R887A rescues of Doxycycline-treated cl.12 cells with endogenous IRE1α knockdown. c RT-qPCR analysis of IRE1α RNase targets CD59, TGOLN2 (RIDD), and TNFAIP8L1, SNN, and SIX2 (RIDDLE). Ct values for XBP1 in sample shIRE1 cl.1 prior to Tg treatment were >34, precluding ratio calculations and were therefore not plotted. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. A 2-way ANOVA test was used to calculate p-values for CD59 and TGOLN2, and an unpaired t -test for the remaining targets. d Analysis of cell viability by Cell-Titer Glo after Dox treatment for 7 days on Ultra-Low Attachment (ULA) plates. n = 2 biologically independent experiments. Data are presented as mean values ± SEM. e Model depicting IRE1α’s principal modes of endoribonuclease function and their underlying phospho-oligomeric states during ER stress. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Antibodies (Abs) for IRE1α (#3294), TGOLN2 (#95649), AIM2 (#12948), Actin (#5125), and GAPDH (#8884) from Cell Signaling Technology.

Techniques: Western Blot, Variant Assay, Expressing, shRNA, Stable Transfection, Transfection, Transgenic Assay, Mutagenesis, Knockdown, Quantitative RT-PCR

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet: CK1G3 localizes to cytoplasmic punctate compartments Intracellular localization of HA-CK1G3. CK1G3 KO cells expressing HA-CK1G3 were immunostained with HA antibody and antibodies against various organelle markers: LBPA (late endosomes), EEA1 (early endosomes), Lamp2 (lysosomes), Hrs (exosomes), VAP (ER), TGN46 (TGN), Rab11a (recycling endosomes and Rab11a-positive secretory vesicles), catalase (peroxisomes), TOMM20 (mitochondria), caveolin (caveolae), and lipid dye II (lipid droplet). For the visualization of lipid droplets, cells were pre-incubated in a medium containing 100 μM oleic acid for 24 h before fixation. The scale bars indicated in microscopy images represent 10 μm.

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Expressing, Incubation, Microscopy

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet: Compartmentalization-dependent functional control of CK1Gs After newly synthesized as a cytosolic protein, CK1G presumably binds to the Golgi-membrane via the electrostatic interaction between its C -terminal basic amino acids stretch and the Golgi localizing PtdIns(4)P (see also the text) and subsequently palmitoylated by the Golgi-residing palmitoyl acyltransferase (PAT). Palmitoylated CK1G is largely distributed to post-Golgi compartments and only partly to the Golgi apparatus, where SM synthase 1 (SMS1) is localized. CERT is associated with the ER via VAP-binding and with the Golgi via PtdIns(4)P-binding. It remains elusive whether palmitoylated CK1Gs are recycled among the distal- and post-Golgi compartments or eventually directed to lysosomes (for degradation) or exosomes (for secretion). The Golgi-distributed CK1G is spatially limited to interact only with CERT recruited to the ER-Golgi contact zone. In contrast, the C -terminus deleted CK1Gs are distributed throughout the cytosol and can phosphorylate CERT anywhere in the cells once the priming phosphorylation of CERT S132 by PKD occurs.

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Functional Assay, Control, Synthesized, Membrane, Binding Assay, Phospho-proteomics

Journal: iScience

Article Title: Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

doi: 10.1016/j.isci.2022.104624

Figure Lengend Snippet:

Article Snippet: Anti-HA antibody (#3F10) was purchased from Roche Diagnostics; anti-CERT (#ab72536) from Abcam; chicken antibody against VAP ( ) anti-TGOLN2/TGN46 (#A304-434A) from Bethyl Laboratories; anti-TOMM20 (#WH0009804M1) from Sigma-Aldrich; anti-Lamp2 (#sc-18822) from Santa Cruz; anti-LBPA (#z-PLBPA) from Echelon Bioscience; anti-EEA1 (#610457) from BD Transduction Laboratories; anti-catalase (#D4P7B) and anti-Rab11a (#2413) from Cell Signaling Technology; anti-Hrs (#10390-1-AP) from Proteintech; anti-GFP (#04404-84) from Nacalai Tesque, and anti-GAPDH (016-25523) from Fujifilm Wako Pure Chemical Corporation.

Techniques: Transduction, Recombinant, Protease Inhibitor, Software

Journal: Journal of Cell Science

Article Title: COMMD1 and PtdIns(4,5)P 2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

doi: 10.1242/jcs.231753

Figure Lengend Snippet: Colocalization of ATP7B with TGN46 or LAMP1. Cells were left untreated (basal medium), treated with 10 μM TTM (low copper) or treated with 10, 100 or 200 μM CuCl2. (A,B) Merged images show ATP7B in green, TGN46 (A) or LAMP1 (B) in magenta and the nucleus in blue; pixel overlap is shown in white. (C-H) 3D colocalization analysis produced M1, M2 and Pearson correlation coefficients for ATP7B and TGN46 (C-E) or LAMP1 (F-H). Values were calculated for each cell and plotted as mean±s.d., with contour plots for visualization of distribution. All data points are shown. Dunnett's method was used to compare treated and untreated cells (MEM); *P<0.05.

Article Snippet: Primary antibodies and concentrations used for immunofluorescence microscopy were as follows: 1:750 rabbit anti-ATP7B (Abcam, Cambridge, MA), 1:1000 mouse anti-LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa), 1:300 sheep anti-TGN46 (Acris Antibodies, San Diego, CA), 1:300 mouse anti-Golgin-97 (Invitrogen, Carlsbad, CA), 1:500 goat anti-VPS35 (Abcam, Cambridge, MA), 1:500 rat anti-HA (Roche Diagnostics, Indianapolis, IN), 1:500 rat anti-C-Myc (Bio-Rad, Hercules, CA), mouse anti-COMMD1 (Novus Biologicals, Littleton, CA), 1:1000 mouse ant-EEA1 1F8-S (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:4000 rabbit anti-LDLR (Invitrogen, Carlsbad, CA).

Techniques: Produced

Journal: Journal of Cell Science

Article Title: COMMD1 and PtdIns(4,5)P 2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

doi: 10.1242/jcs.231753

Figure Lengend Snippet: Colocalization of ATP7B with TGN46 or LAMP1 in cells with reduced COMMD1 expression. Cells were transfected with COMMD1 (siCD1) or nontarget (control) siRNA and treated with TTM (low copper) or 200 μM CuCl2 and cycloheximide for the last hour. (A-C) 3D colocalization analysis of ATP7B with TGN46. (D-F) 3D colocalization analysis of ATP7B with LAMP1. The M1, M2 and Pearson coefficients were calculated for each cell and plotted as mean±s.d., with contour plots for visualization of distribution. All data points are shown. Student's t-test was used to compare COMMD1 knockdown with the control for each copper treatment; *P<0.05, **P<0.005.

Article Snippet: Primary antibodies and concentrations used for immunofluorescence microscopy were as follows: 1:750 rabbit anti-ATP7B (Abcam, Cambridge, MA), 1:1000 mouse anti-LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa), 1:300 sheep anti-TGN46 (Acris Antibodies, San Diego, CA), 1:300 mouse anti-Golgin-97 (Invitrogen, Carlsbad, CA), 1:500 goat anti-VPS35 (Abcam, Cambridge, MA), 1:500 rat anti-HA (Roche Diagnostics, Indianapolis, IN), 1:500 rat anti-C-Myc (Bio-Rad, Hercules, CA), mouse anti-COMMD1 (Novus Biologicals, Littleton, CA), 1:1000 mouse ant-EEA1 1F8-S (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:4000 rabbit anti-LDLR (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Transfection

Journal: Journal of Cell Science

Article Title: COMMD1 and PtdIns(4,5)P 2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

doi: 10.1242/jcs.231753

Figure Lengend Snippet: Colocalization of ATP7B with TGN46 or LAMP1 in cells with reduced COMMD1 expression. Cells were transfected with COMMD1 (siCD1) or nontarget (control) siRNA and treated with TTM (low copper) or 200 μM CuCl2 and cycloheximide with the addition of MG132 and chloroquine (CLQ) for the last hour. (A-C) 3D colocalization analysis of ATP7B with TGN46. (D-F) 3D colocalization analysis of ATP7B with LAMP1. The M1, M2 and Pearson coefficients were calculated for each cell and plotted as mean±s.d., with contour plots for visualization of distribution. All data points are shown. Student's t-test was used to compare COMMD1 knockdown with the control for each copper treatment; *P<0.05.

Article Snippet: Primary antibodies and concentrations used for immunofluorescence microscopy were as follows: 1:750 rabbit anti-ATP7B (Abcam, Cambridge, MA), 1:1000 mouse anti-LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa), 1:300 sheep anti-TGN46 (Acris Antibodies, San Diego, CA), 1:300 mouse anti-Golgin-97 (Invitrogen, Carlsbad, CA), 1:500 goat anti-VPS35 (Abcam, Cambridge, MA), 1:500 rat anti-HA (Roche Diagnostics, Indianapolis, IN), 1:500 rat anti-C-Myc (Bio-Rad, Hercules, CA), mouse anti-COMMD1 (Novus Biologicals, Littleton, CA), 1:1000 mouse ant-EEA1 1F8-S (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:4000 rabbit anti-LDLR (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Transfection

Journal: Journal of Cell Science

Article Title: COMMD1 and PtdIns(4,5)P 2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

doi: 10.1242/jcs.231753

Figure Lengend Snippet: Colocalization of ATP7B with TGN46 or LAMP1 in cells with reduced COMMD1 misexpression. Cells were transfected with one of three COMMD1 variants: GFP-COMMD1, GFP-COMMD1 T174M and GFP-COMMD1 K167/173E. They were then treated with TTM (low copper) or 200 μM CuCl2 for 9 h with cycloheximide added for the last hour. (A-C) 3D colocalization analysis of ATP7B with TGN46. (D-F) 3D colocalization analysis of ATP7B with LAMP1. The M1, M2 and Pearson coefficients were calculated for each cell and plotted as mean±s.d., with contour plots for visualization of distribution. All data points are shown. Dunnett's method was used to compare COMMD1 variants with the wild type; *P<0.05, **P<0.005.

Article Snippet: Primary antibodies and concentrations used for immunofluorescence microscopy were as follows: 1:750 rabbit anti-ATP7B (Abcam, Cambridge, MA), 1:1000 mouse anti-LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa), 1:300 sheep anti-TGN46 (Acris Antibodies, San Diego, CA), 1:300 mouse anti-Golgin-97 (Invitrogen, Carlsbad, CA), 1:500 goat anti-VPS35 (Abcam, Cambridge, MA), 1:500 rat anti-HA (Roche Diagnostics, Indianapolis, IN), 1:500 rat anti-C-Myc (Bio-Rad, Hercules, CA), mouse anti-COMMD1 (Novus Biologicals, Littleton, CA), 1:1000 mouse ant-EEA1 1F8-S (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:4000 rabbit anti-LDLR (Invitrogen, Carlsbad, CA).

Techniques: Transfection

Journal: Journal of Cell Science

Article Title: COMMD1 and PtdIns(4,5)P 2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

doi: 10.1242/jcs.231753

Figure Lengend Snippet: Colocalization of ATP7B with TGN46 or LAMP1 in response to PtdIns(4,5)P2 modulation. HepG2 cells were transfected with the PH domain or ArfQ67L then treated with TTM (low copper) or 200 μM CuCl2 for 9 h with cycloheximide added for the last hour. (A-C) 3D colocalization analysis of ATP7B with TGN46. (D-F) 3D colocalization analysis of ATP7B with LAMP1. The M1, M2 and Pearson coefficients were calculated for each cell and plotted as mean±s.d., with contour plots for visualization of distribution. All data points are shown. Dunnett's method was used to compare cells expressing indicated proteins with control; *P<0.05, **P<0.005.

Article Snippet: Primary antibodies and concentrations used for immunofluorescence microscopy were as follows: 1:750 rabbit anti-ATP7B (Abcam, Cambridge, MA), 1:1000 mouse anti-LAMP1 (Developmental Studies Hybridoma Bank, Iowa City, Iowa), 1:300 sheep anti-TGN46 (Acris Antibodies, San Diego, CA), 1:300 mouse anti-Golgin-97 (Invitrogen, Carlsbad, CA), 1:500 goat anti-VPS35 (Abcam, Cambridge, MA), 1:500 rat anti-HA (Roche Diagnostics, Indianapolis, IN), 1:500 rat anti-C-Myc (Bio-Rad, Hercules, CA), mouse anti-COMMD1 (Novus Biologicals, Littleton, CA), 1:1000 mouse ant-EEA1 1F8-S (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:4000 rabbit anti-LDLR (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Expressing

Journal: Neuropathology

Article Title: Distinctive features of degenerating Purkinje cells in spinocerebellar ataxia type 31

doi: 10.1111/neup.12090

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: Trans-Golgi network protein 2 (TGOLN2) , Mouse/monoclonal (clone 2F11) , 1:1000 , Autoclaving , Abnova, Taiwan.

Techniques: Binding Assay

Journal: Neuropathology

Article Title: Distinctive features of degenerating Purkinje cells in spinocerebellar ataxia type 31

doi: 10.1111/neup.12090

Figure Lengend Snippet: Immunohistochemistry for trans-Golgi network protein 2 (TGOLN2). (A) Granular appearance of a Purkinje cell from a control subject (arrowheads). (B) Normal-sized Purkinje cells without the halo-like amorphous materials from case 1 with spinocerebellar ataxia type 31 (SCA31) show the relative preservation of the Golgi apparatus (arrowheads). (C and D) Purkinje cells with the halo-like amorphous materials (arrows) from case 1 (C) and 2 (D) with SCA31 show severely fragmented and a reduced amount of the Golgi apparatus (arrowheads). (E and F) Shrunken Purkinje cells without the halo-like amorphous materials from cases 1 (E) and 2 (F) with SCA31 show reduced, but not fragmented Golgi apparatus (arrowheads). Bars; 20 μm.

Article Snippet: Trans-Golgi network protein 2 (TGOLN2) , Mouse/monoclonal (clone 2F11) , 1:1000 , Autoclaving , Abnova, Taiwan.

Techniques: Immunohistochemistry, Preserving

Journal: The EMBO Journal

Article Title: Non-autophagic Golgi-LC3 lipidation facilitates TFE3 stress response against Golgi dysfunction

doi: 10.1038/s44318-024-00233-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit monoclonal anti-TGOLN2 , Abclonal , Cat # A19618;.

Techniques: Recombinant, Sequencing, Virus, Reverse Transcription, SYBR Green Assay, Protease Inhibitor, Software