Journal: Journal of Virology

Article Title: NFAT4 Is Required for JC Virus Infection of Glial Cells

doi: 10.1128/jvi.01456-06

Figure Lengend Snippet: FIG. 2. NFAT4 is functionally active in glial cells. (a) NFAT expression in SVG-A and U-87MG whole-cell lysates. NFATs 1 to 4 were detected using antibodies (NFATc1 7A6, NFATc2 4G6-G5, NFATc3 F-1, and NFATc4 H-74; Santa Cruz Biotech Inc.) diluted 1:200. Ramos cell lysates were used as a positive control. (b) Luciferase reporter gene assays using an NFAT-responsive reporter construct were used to compare NFAT activity in SVG-A cells and U-87MG cells in the presence or absence of 292 mg/liter glutamate. A control construct that lacked the NFAT binding site was used to measure basal transcriptional activity. (c) The NFAT reporter construct was cotransfected with either a control construct ( SV40 T-Ag) or a construct expressing the SV40 large T-Ag ( SV40 T-Ag).

Article Snippet: Ramos cell lysates were purchased from Santa Cruz Biotech Inc. Whole-cell extracts were separated on Tris-HCl-ready gels (Bio-Rad).

Techniques: Expressing, Positive Control, Luciferase, Construct, Activity Assay, Control, Binding Assay

Journal: Human Molecular Genetics

Article Title: Expression of Ripk1 and DAM genes correlates with severity and progression of Krabbe disease

doi: 10.1093/hmg/ddab159

Figure Lengend Snippet: Autophagosomal/lysosomal dysfunction accompanies Ripk1 expression in Krabbe and SD. Autophagy was studied by ( A ) RT-qPCR of total RNA in spinal cord at three stages of disease in twi-2J and at the HEP in SD, with markers Atg5 (Autophagy related 5), LC3B (Microtubule-associated protein 1 light chain 3 beta), and Sqstm1 (Sequestosome 1 codes for p62); ( B ) Immunoblot of LC3B and p62 in twi-2J spinal cord at different ages. Protein extracts from Neuro2a chloroquine-treated cells were used as positive control of upregulated autophagy; ( C ) Immunoblot to LC3B and p62 and densitometry in twi-2J brain stem at HEP. Seventy-five microgram protein homogenate was loaded per lane. Blots were stripped of first antibody and re-probed with subsequent antibodies. Fluorescent IHC staining of p62 in twi-2J sciatic nerve and brain ( D ), and SD spinal cord at HEP ( E ). Dual IHC staining of ubiquitin1 and p62 in twi-2J spinal cord at HEP ( F ) and SD ( G ). ( H ) Dual IHC staining of ubiquitin1 and LC3B in twi-2J spinal cord at HEP. Nuclear stain: DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride), mutant (mut), wild type (wt). Student’s t -test; * P ≤ 0.05; * * P ≤ 0.01.

Article Snippet: For polyacrylamide-gel electrophoresis and immunoblotting, after reduction and denaturation in sodium dodecyl sulphate and 4% β-mercaptoethanol, 5–200 μg of protein extracts from tissue or from cells 293T transfected with a plasmid expressing mouse caspase-8 (OriGene Technologies #MC200404) and Neuro2a chloroquine-treated (Novus Biologicals #NBP2-49688) were heated at 90°C and run in 4–15% linear gradient gels (161–1122; Bio-Rad) or in 8, 10, 12 and 15% gels made in-house, proteins were transferred onto 0.45 μm PVDF membranes (Millipore #IPV00010).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Positive Control, Immunohistochemistry, Staining, Mutagenesis

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a A UBOX5 chimeric construct (UBOX5-UBD) where the UBOX5 open reading frame was fused by a flexible linker to a FLAG-tagged ubiquitin binding domain was generated. FLAG-tagged UBOX5 (3 lanes on the right) or FLAG-tagged UBOX5-UBD construct (4 lanes on the left) and HA-tagged ubiquitin was co-transfected into HEK293 cells. After the first immunoprecipitation of lysates by anti-FLAG antibody, 20% of eluates was kept for analysis. Eluate from the UBOX5-UBD was further immunoprecipitated with anti-HA antibody to enrich for ubiquitinated proteins. To serve as antibody specificity control (negative controls), lysates were mock immunoprecipitated with mouse immunoglobulin. Immunoblotting of BIP was performed on inputs and eluates as indicated. Bands corresponding to ubiquitinated BIP and ubiquitinated UBOX5-UBD chimeric protein are indicated by vertical lines, while the unmodified proteins are indicated by arrows on the right. Positions of the molecular weight markers are indicated by arrows on the left. IB: FLAG and IB:HA are shown in Supplementary Fig. . The experiment was repeated independently 3 times. Source data are provided as a Source data file. b MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 h later, cells were treated with 0.7 uM Tharpsigargin for 16 h, and then further treated with MG132 (a proteasomal inhibitor) for 6 h as indicated. Cells were then harvested and a MYC immunoprecipitation was performed on the input lysates. Eluates were immunoblotted with antibodies against HA to assess the extent of BIP ubiquitination. The membrane was then stripped and a MYC immunoblot was performed to assess immunoprecipitation efficiency. Ubiquitination of BIP was only observed when UBOX5 was expressed. The degree of ubiquitination of BIP did not appear to differ with the addition of MG132, a proteasomal inhibitor, suggesting that the ubiquitinated BIP was not degraded by the proteasome pathway. The experiment was repeated independently 3 times. Source data are provided as a Source data file. (c) UBOX5 or its empty vector contains a GFP open reading frame, which allows for assessment of transfection efficiency by assessing GFP abundance in input lysates. Human UBOX5 immunoblots were used to verify expression of UBOX5. GAPDH was used as loading control. The experiment was repeated independently 3 times. Source data are provided as a Source data file.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Construct, Ubiquitin Proteomics, Binding Assay, Generated, Transfection, Immunoprecipitation, Control, Western Blot, Molecular Weight, Plasmid Preparation, Membrane, Expressing

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a , b UBOX5 and BIP are both induced by ER stress. a NIH3T3 cells were treated with the indicated ER stress inducers tunicamycin (Tu) and thapsigargin (Tg). (Upper) Endogenous UBOX5 mRNA abundance was quantified by qPCR in biological triplicates, normalised against mouse beta-actin transcript. Relative fold change of transcript is reported against control DMSO treatment. Error bars represent standard deviation. ( b , Top) Protein abundance of endogenous mouse UBOX5 in ER-stressed NIH3T3 cells. Beta-actin was used as loading control. Of note, UBOX5 mRNA and protein is induced in response to ER stress. ( b , Bottom): Densitometric quantitation of UBOX5 bands. UBOX5 band intensity of each sample is normalised to corresponding beta-actin intensity. The amount of TG or TU used is indicated on the x -axis. P -values were generated from two-sided Welch’s t -test. Error bars represent standard deviation. Source data are provided as a Source data file. c Cellular Localization of UBOX5: Immunoblots of HEK293 cells transiently transfected with UBOX5 expression plasmid and treated with 0.7 uM Thapsigargin for 16 h. Cellular fractions are indicated above. Whole cell lysates were fractionated into cytoplasmic, endoplastic reticulum (ER) fractions and nuclear fractions by stepwise centrifugation. Indicated antibodies are shown. Positions of molecular weight markers are indicated on the left with arrows. GFP is used as transfection control, Calnexin is used as fractionation control for ER and nuclear fraction; Histone H2B is used as fractionation control for nuclear fraction. GAPDH is used as fractionation control for cytoplasmic and nuclear fractions. The experiment was repeated independently 3 times. Source data are provided as a Source data file. d , e The ability of UBOX5 to ubiquitinate BIP is not dependent on cellular stress. d MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 hours later, cells were treated with 0.7 μM Thapsigargin (TG) or DMSO for 16 h. TG is a known inducer of endoplasmic reticulum stress. Cells were then harvested and a MYC immunoprecipitation was performed on the input lysates. Eluates were immunoblotted with antibodies against HA to assess the extent of BIP ubiquitination. e UBOX5 or its empty vector contains a GFP open reading frame, which allows for assessment of transfection efficiency by assessing GFP abundance in input lysates. GAPDH was used as loading control.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Control, Standard Deviation, Quantitative Proteomics, Quantitation Assay, Generated, Western Blot, Transfection, Expressing, Plasmid Preparation, Centrifugation, Molecular Weight, Fractionation, Ubiquitin Proteomics, Immunoprecipitation

Journal: Nature Communications

Article Title: Functionally deficient UBOX5 variants and primary angle-closure glaucoma

doi: 10.1038/s41467-025-62775-x

Figure Lengend Snippet: a Wild-type UBOX5 increases the half-life of BIP in the presence of thapsigargin-induced ER stress. Top left panel: Pulse chase of BIP with or without expression of UBOX5 in HEK293 cells. HEK 293 cells were transfected with UBOX5 or empty vector. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 hours, and cycloheximde (150 mM) was added. Cells were collected at indicated time points for immunoblotting. GAPDH was used as loading control. Positions of molecular weight standards are indicated on the left. Top right panel: The same experiment repeated with Tharpsigargin substituted with DMSO carrier control. Bottom panels: Quantitation of BIP band intensities normalized to GAPDH intensity for every indicated time point. Separate graphs were shown for cells treated with Tharpsigargin (Left) or DMSO control (Right). Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Data are presented as mean values +/- standard deviation. Error bars indicate standard deviation; 3 biological replicates were used for quantitation. Source data are provided as a Source data file. b Variant UBOX5 and their effects on the half-life of BIP. Top: Pulse chase of BIP in the presence of wildtype UBOX5 or variant UBOX5 (D33N, K291R, R301Q, and S465C) in HEK293 cells. HEK 293 cells were transfected with wildtype UBOX or indicated variants. 24 h later, cells were treated with a pulse of 0.25 μM Tharpsigargin for 2 h, and cycloheximde (150 mM) was added at time = 0. Cells were then subsequently collected at indicated time points (in hours) for immunoblotting. GAPDH was used as loading control. UBOX5 expression was verified as indicated. GFP, expressed from a separate locus in the vector used, was also used to verify success of transfection. Bottom: Densitometric quantitation of BIP band intensities in cells expressing indicated UBOX5 variants, normalized to GAPDH intensity for every time point on the same blot. Intensities are shown as fold changes compared to normalized BIP intensity at t = 0 of pulse chase. Three biological replicates were analyzed. Error bars represent standard deviation. Source data are provided as a Source data file.

Article Snippet: HEK293 cells were transiently transfected with 1.8 μg wild-type UBOX5 expression plasmid per 10 cm dish and treated with 0.7 μM Thapsigargin (#sc-24017, Santa Cruz) for 16 h. Cells were then collected and lysed in Subcellular (SF) Buffer (250 mM Sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, PPI cocktail), and left to rotate on a rotary shaker for 30 min at 4 °C.

Techniques: Pulse Chase, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Molecular Weight, Quantitation Assay, Standard Deviation, Variant Assay