Journal:

Article Title: Specificity and Prevalence of Natural Bovine Anti-Alpha Galactosyl (Gal?1-6Glc or Gal?1-6Gal) Antibodies

doi:

Figure Lengend Snippet: Measurement and comparison of anti-alpha galactosyl IgA, IgG1, and IgG2 responses. The anti-alpha galactosyl IgA, IgG1, and IgG2 antibodies were measured using class- or isotype-specific antibodies conjugated to horseradish peroxidase as described in Materials and Methods. All sera were diluted 1:40. The serum sample numbers correspond to those in Fig. ​Fig.4.4. Panels: A, Anti-alpha galactosyl IgA, IgG1, and IgG2 levels expressed as direct OD values; B, Anti-alpha galactosyl IgA, IgG1, and IgG2 levels expressed as percentages.

Article Snippet: Anti-alpha galactosyl IgG1, IgG2, and IgA were measured by an indirect ELISA using affinity-purified anti-bovine IgA, IgG1, and IgG2 antibodies conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, Tex.).

Techniques:

Journal: bioRxiv

Article Title: Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

doi: 10.1101/349407

Figure Lengend Snippet: (A) GFP u* degradation requires catalytically active UBE3C. Top: Wild type or catalytically inactive mutant UBE3C (UBE3C C1051A ) was expressed in UBE3C KO cells and GFP u* turnover was assessed following inhibition of protein synthesis with emetine for the indicated times. Bottom: Quantification of GFP u* turnover. Data are representative of two independent experiments. (B) UBE3C is required for conjugation of high molecular weight Ub conjugates to GFP u* . Dox-induced GFP u* reporter cells were treated with the indicated inhibitors for 4 hr. PolyUb conjugates were affinity captured from cell lysates using immobilized Halo-UBQLN1 UBA and analyzed by immunoblotting with an anti-GFP antibody. (C) Conjugation of high molecular weight Ub chains to GFP u* requires catalytically active UBE3C. PolyUb conjugates were affinity purified as in (B) from wild type or UBE3C K0 cells expressing the indicated rescue constructs. Ubiquitylated GFP u* was visualized by SDS-PAGE and immunoblotting with an anti-GFP antibody. (D) Schematic diagram for the analysis in (E). Halo-TRABID NZF1 binds selectively to K29-linked Ub chains. (E) PolyUb conjugates were affinity captured from GFP u* cell lysates with immobilized Halo-TRABID NZF1 and the presence of K29 Ub linkages on GFP u* was assessed by incubating with the catalytic domain of vOTU, which does not hydrolyze K27 or K29 linkages, or with the core catalytic domain of the nonspecific DUB Usp2 (Usp2cc). Ubiquitylated species were separated by SDS-PAGE and GFP u* was visualized by immunoblotting with an anti-GFP antibody. Arrows indicate polyUb GFP u* species that are resistant to deubiquitylation by vOTU. See also .

Article Snippet: The primary antibodies used in this study are rabbit α-UBE2G2 mAb (Abcam ab174296; 1:2,500); rabbit α-AUP1 pAb (Proteintech 13726-1-AP, 1:1,000); rabbit α-UBE3C pAb (Bethyl Laboratories A304-122A, 1:1,000); mouse α-UBE2D3 mAb (Abcam ab58251; 1:2,000); mouse α-GFP JL8 mAb (Takara 632381; 1:2,000); rabbit α-GFP mAb (Cell Signaling Technology 2956; 1:1,000); mouse α-PDI mAb (Enzo Life Sciences ADI-SPA-891; 1:1000); rabbit α-VCP pAb (Novus Biologicals NB100-1558; 1:10,000); rabbit α-GAPDH pAb (Millipore ABS16; 1:20,000); mouse α-GAPDH mAb (Proteintech 60004-1-Ig; 1:20,000); rabbit α-alpha tubulin pAb (Abcam ab15246; 1:10,000); mouse α-RTA mAb (BioRad Laboratories MCA2865; 1:2,000); mouse α-CD147 mAb (Santa Cruz Biotechnology Inc. sc-25273; 1:1,000); rabbit α-SEL1L rAb (custom antibody; 1:1,000); rabbit α-Ub pAb (Cell Signaling Technology 3933S; 1:1,000); mouse α-Ub FK2 mAb (Millipore Sigma 04-263; 1:1,000); rabbit α-K48 polyUb, clone Apu2 mAb (Millipore Sigma 05-1307; 1:5,000); human α-K48 polyUb, clone Apu2.07 (Genentech; 0.2 μg/mL); human α-K11 polyUb, clone 2A3/2E6 (Genentech; 0.5 μg/mL); human α-K11/K48 bispecific polyUb (Genentech; 0.2 μg/mL); human α-K11/gD bispecific control (Genentech; 0.2 μg/mL); human α-K48/gD bispecific control (Genentech; 0.2 μg/mL).

Techniques: Mutagenesis, Inhibition, Conjugation Assay, High Molecular Weight, Western Blot, Affinity Purification, Expressing, Construct, SDS Page

Journal: bioRxiv

Article Title: Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

doi: 10.1101/349407

Figure Lengend Snippet: (Left) The glycosylated luminal ERAD substrate A1AT NHK -GFP requires ER mannosidases and glycan recognition machinery, the membrane-embedded HRD1 dislocation complex, and ER-embedded UPS components for ER exit and delivery to the proteasome, exemplifying the canonical HRD1-dependent ERAD-L degradation pathway. Substrate-specific heterogeneity in the HRD1-dependent pathway is revealed by the luminal glycosylated protein GFP-RTA E177Q , which can engage luminal recognition factors for slow delivery to the HRD1 complex or can be rapidly dislocated and degraded by bypassing upstream quality control machinery. Dislocated ERAD substrates are modified with K48 and K11 Ub linkages and targeted for destruction by cytosolic proteasomes. (Middle) The ERAD-M substrate INSIG1-GFP utilizes GP78 for ER dislocation and ubiquitylation. Efficient delivery of this highly hydrophobic substrate to cytosolic degradation machinery may be facilitated by conjugation of K11/K48 chains that increase affinity for p97/VCP and the proteasome. (Right) Efficient degradation of the ERAD-C substrate GFP u* requires conjugation of heterotypic K48/K29 chains via the ER-embedded E3 ligase TRC8 and the cytosolic E3 UBE3C, which may facilitate interactions with proteasome shuttling factors or increase substrate processing at the proteasome.

Article Snippet: The primary antibodies used in this study are rabbit α-UBE2G2 mAb (Abcam ab174296; 1:2,500); rabbit α-AUP1 pAb (Proteintech 13726-1-AP, 1:1,000); rabbit α-UBE3C pAb (Bethyl Laboratories A304-122A, 1:1,000); mouse α-UBE2D3 mAb (Abcam ab58251; 1:2,000); mouse α-GFP JL8 mAb (Takara 632381; 1:2,000); rabbit α-GFP mAb (Cell Signaling Technology 2956; 1:1,000); mouse α-PDI mAb (Enzo Life Sciences ADI-SPA-891; 1:1000); rabbit α-VCP pAb (Novus Biologicals NB100-1558; 1:10,000); rabbit α-GAPDH pAb (Millipore ABS16; 1:20,000); mouse α-GAPDH mAb (Proteintech 60004-1-Ig; 1:20,000); rabbit α-alpha tubulin pAb (Abcam ab15246; 1:10,000); mouse α-RTA mAb (BioRad Laboratories MCA2865; 1:2,000); mouse α-CD147 mAb (Santa Cruz Biotechnology Inc. sc-25273; 1:1,000); rabbit α-SEL1L rAb (custom antibody; 1:1,000); rabbit α-Ub pAb (Cell Signaling Technology 3933S; 1:1,000); mouse α-Ub FK2 mAb (Millipore Sigma 04-263; 1:1,000); rabbit α-K48 polyUb, clone Apu2 mAb (Millipore Sigma 05-1307; 1:5,000); human α-K48 polyUb, clone Apu2.07 (Genentech; 0.2 μg/mL); human α-K11 polyUb, clone 2A3/2E6 (Genentech; 0.5 μg/mL); human α-K11/K48 bispecific polyUb (Genentech; 0.2 μg/mL); human α-K11/gD bispecific control (Genentech; 0.2 μg/mL); human α-K48/gD bispecific control (Genentech; 0.2 μg/mL).

Techniques: Glycoproteomics, Membrane, Control, Modification, Conjugation Assay

Journal: bioRxiv

Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2

doi: 10.1101/457176

Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05-636), Rad51 (Cosmo Bio Co. Ltd, 70-001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791) were used.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) qRT-PCR analysis of Brg1, Cdx2, and Oct4 transcripts in Brg1 KD 8-cell embryos, morulae, and blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control embryos at each stage; black line = 1. Asterisk denotes significant difference between Brg1 KD and control blastocysts (p<0.05). (B) ICC analysis of Oct4 and Cdx2 expression in Brg1 KD 8-cell embryos, morulae, and blastocysts. Nuclei were counter stained with DAPI (blue). (C) Brg1 represses Oct4 expression in a dose dependent manner. One-cell embryos were injected with 0 µM (control), 0.1 µM, 1 µM, or 100 µM Brg1 siRNA and cultured to the blastocyst stage. Real-time qPCR was used to analyze the levels of Brg1 and Oct4 transcripts. Data were normalized to Ubtf and are relative to control blastocysts; dashed line = 1.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Quantitative RT-PCR, Control, Expressing, Staining, Injection, Cell Culture

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) ICC analysis of Oct4 and Cdx2 in Brg1 KD and control blastocysts. In control blastocysts (a–d) Oct4 expression (green) is restricted to the ICM and is largely absent in the Cdx2-positive (red) trophectoderm. In contrast, in Brg1 KD blastocysts (e–h) Oct4 is widely expressed in both the ICM and cdx2-positive (yellow) trophectoderm. Arrowheads denote co-expression of Oct4 and Cdx2. Nuclei were counter stained with DAPI (blue). (B) Quantification of the average number of cells in Brg1 KD and control blastocysts expressing Oct4, Cdx2, and Oct4 & Cdx2 (double expression). Asterisks denote statistical significance (p<0.05) between Brg1 KD and control blastocysts. A total of 25 Brg1 KD blastocysts and 15 control blastocysts were analyzed.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Control, Expressing, Staining

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) Combined depletion of Brg1 and Cdx2 augments Oct4 expression in blastocysts. qRT-PCR analysis of Oct4 transcripts in Brg1 KD blastocysts, Cdx2 KD blastocysts, and Brg1 & Cdx2 double KD blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control blastocysts; dashed line = 1. Different letters denote statistical significance in Oct4 transcripts (p<0.05). These experiments were replicated using a total of 5 biological replicates. (B) Co-immunoprecipitation and western blot analysis of Brg1 and Cdx2 in TS cells. Brg1 was immunoprecipitated using a rabbit anti-serum. Recovery of Cdx2 was measured by western blot analysis. Cdx2 is enriched in the Brg1 IP samples and not in the control IgG samples. This assay was repeated a total of 4 times using different batches of TS cells. (C) Confocal immunofluorescence analysis of Brg1 and Cdx2 in blastocysts. Co-localization of endogenous Brg1 and Cdx2 in trophectoderm nuclei was determined using specific antibodies for Brg1 and Cdx2. Nuclei were counterstained with DAPI. White box represents magnified region in bottom panel. Arrow denotes blastocyst ICM. (D) Confirmation of Flag-Cdx2 expression in induced ES cells. Western blot and immunofluorescence analysis of Flag-Cdx2 expression at 24 hours following removal of doxycycline. (E) ChIP analysis of Brg1 and Cdx2 binding to the Oct4 promoter in Cdx2-inducible ES cells. qRT-PCR was used to determine the relative enrichment of Brg1 and Flag-Cdx2 at the Oct4 ARE versus an intergenic region in uninduced and induced ES cell extracts. A non-specific rabbit IgG was included as a negative control. Data were normalized to 1% input DNA. Asterisks denote significant differences between uninduced and induced samples (p<0.05). These experiments were replicated 3 to 4 times using two different batches of Cdx2-inducible ES cell extracts.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot, Immunofluorescence, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Schematic diagram of Oct4 regulation in blastocysts. In the trophectoderm Brg1 is recruited to the ARE of the Oct4 promoter via Cdx2. Once at the Oct4 promoter Brg1 and Cdx2 facilitate recruitment of additional co-repressors to repress transcription.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques:

Journal:

Article Title: Immunodiagnostic Identification of Dairy Cows Infected with Prototheca zopfii at Various Clinical Stages and Discrimination between Infected and Uninfected Cows

doi: 10.1128/JCM.39.2.539-543.2001

Figure Lengend Snippet: Immunoblot of a preparative SDS-PAGE analysis of whole-cell antigen of P. zopfii to detect specific IgG antibodies in serum and IgA antibodies in whey. Serum and whey were obtained from culture-positive (+) and culture-negative (−) dairy cows. Immunogenic components of 30 and 32 kDa are indicated by arrows.

Article Snippet: The following affinity-purified polyclonal monospecific peroxidase-conjugated conjugates were used: anti-bovine IgG, anti-bovine IgG1, anti-bovine IgA, or anti-bovine IgM (Bethyl Laboratories, Inc., Montgomery, Tex.).

Techniques: Western Blot, SDS Page

Journal:

Article Title: Immunodiagnostic Identification of Dairy Cows Infected with Prototheca zopfii at Various Clinical Stages and Discrimination between Infected and Uninfected Cows

doi: 10.1128/JCM.39.2.539-543.2001

Figure Lengend Snippet: ELISA activity of IgG (dark gray boxes) in serum (A) and of IgA (dotted boxes) (B) and IgG1 (white boxes) (C) in whey of cows with various clinical stages of P. zopfii infection. A significant discrimination (P < 0.05) of acutely infected culture-positive versus chronically infected culture-positive animals is indicated by asterisks. ⧫, Significant discrimination (P < 0.05) of chronically infected culture-positive versus chronically infected culture-negative animals. A significant discrimination (P < 0.05) of chronically infected culture-positive versus noninfected cows is indicated by a dagger symbol. The cutoff value for positive test results is pointed by the dotted horizontal line.

Article Snippet: The following affinity-purified polyclonal monospecific peroxidase-conjugated conjugates were used: anti-bovine IgG, anti-bovine IgG1, anti-bovine IgA, or anti-bovine IgM (Bethyl Laboratories, Inc., Montgomery, Tex.).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Infection