Journal: Brain, Behavior, & Immunity - Health

Article Title: The origin and nature of the complex autoantibody profile in cerebrospinal fluid

doi: 10.1016/j.bbih.2019.100032

Figure Lengend Snippet: Comparison of aAB reactivity in plasma and CSF obtained from female and male non-delirium control subjects . Blocks of 75 proteins on human protein microarrays that were probed with diluted plasma and CSF were randomly selected to generate plasma (red line) and CSF (blue line) aAB profiles derived from an 84 y/o female non-delirium control subject (A) and an 84 y/o male non-delirium control subject (B). When the histograms representing the RFUs from each of the 75 protein targets in plasma and CSF are aligned, the observed pattern of RFUs in plasma and CSF is closely matched, and neither advanced age nor gender was found to influence this relationship.

Article Snippet: Invitrogen’s ProtoArray v5.1 Human Protein Microarrays (Cat. No. PAH0525020, Invitrogen, Carlsbad, CA, USA), each containing 9486 unique human protein antigens ( www.invitrogen.com/protoarray ), were used for aAB detection.

Techniques: Derivative Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: The origin and nature of the complex autoantibody profile in cerebrospinal fluid

doi: 10.1016/j.bbih.2019.100032

Figure Lengend Snippet: Fidelity of aAB profiles in a single individual over a period of 9 years . Four different blocks of 50 randomly selected proteins on human protein microarrays that were probed with diluted plasma (red line) and CSF (blue line) showing aAB profiles of a single healthy individual spanning a period of 9 years. aAB profiles remained essentially unchanged over the 9 year period, providing strong evidence that, in the absence of pathology, each individual has a unique and stable baseline aAB profile in the blood that demonstrates a high degree of fidelity over time.

Article Snippet: Invitrogen’s ProtoArray v5.1 Human Protein Microarrays (Cat. No. PAH0525020, Invitrogen, Carlsbad, CA, USA), each containing 9486 unique human protein antigens ( www.invitrogen.com/protoarray ), were used for aAB detection.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: Sera from 51 ICL patients and 25 HCs were screened for autoantibodies using a high-throughput 124 autoantigen microarray platform. (A) Volcano plots of the IgG and IgM autoantibodies, on the left and the right, respectively, displaying –log10(P value) on the y axis versus log2 (average Ab score in ICL samples/average Ab score in HC samples) on the x axis. Each circle represents an autoantibody, highlighting in blue (IgG) or green (IgM) the statistically significant positive autoantibodies between the HC and ICL groups, calculated with the nonparametric Mann-Whitney test with Bonferroni’s correction. Only targets having P < 0.05/122 (with 122 being the number of comparisons) were considered significant and are highlighted. (B) Venn diagram showing antigens recognized by both IgG and IgM (pink) vs. by only IgG (blue) or only IgM (green) autoantibodies. (C) Number of autoantibody targets with Z ≥ 4 for HCs and each subgroup of ICL patients. Z scores for each target were calculated as the number of standard deviations the Ab score was above the mean of the HC Ab score for of each target. Group 1 (open circles, n = 22) corresponds to ICL patients without a diagnosed autoimmune disease and without a positive test for a set of clinical autoantibodies. Group 2 (cyan circles, n = 15) corresponds to patients who tested positive for clinical autoantibodies but did not meet clinical criteria for any specific autoimmune diagnosis. Group 3 (blue circles, n = 14) corresponds to patients who had been diagnosed with 1 or more autoimmune disease. Data were pooled from 2 independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001 by Kruskal-Wallis test and Dunn’s correction for multiple comparisons.

Article Snippet: The sera were then incubated on the Human Protein Microarray v5.1 (Thermo Fisher Scientific) displaying more than 9,000 human proteins.

Techniques: High Throughput Screening Assay, Microarray, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: Sera from ICL patients (n = 34) and HCs (n = 15) were screened for the presence of autoantibodies using the Human Protein Microarray v5.2. Sera were incubated on a microarray that displays over 9,000 full-length purified human proteins in their native conformations. Data were batch corrected, filtered for relative fluorescence units (RFUs) >500 for at least 1 sample for each particular protein, and normalized. The Z score for each target was calculated as the number of standard deviations the signal for a specific target had above the mean of the HCs. (A) Proportion of HC (gray) or ICL patients (blue) that had IgG Abs at Z scores ≥3, ≥4, or ≥5. (B) Number of proteins targeted by IgG Abs with Z score ≥4 from individual HC (gray) or patients’ sera grouped according to autoimmune status, as described in Figure 1C. (C) Percentage of participants (HC, gray; ICL, blue) that shared any of the 2,159 targets found at Z ≥ 4. Data were pooled from 3 independent experiments. **P < 0.01 by Kruskal-Wallis test and Dunn’s correction for multiple comparisons.

Article Snippet: The sera were then incubated on the Human Protein Microarray v5.1 (Thermo Fisher Scientific) displaying more than 9,000 human proteins.

Techniques: Microarray, Incubation, Purification, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A, B) LMBR1L physically interacts with components of the Wnt signaling pathway. (A) Human protein microarray revealed binding between LMBRlL and GSK-3β proteins. A construct expressing both N-terminus FLAG-tagged and C-terminus V5-tagged human LMBR1L was transfected into HEK293T cells and the recombinant protein was purified using anti-FLAG M2 agarose beads. Binding between recombinant human LMBR1L and purified human proteins printed in duplicate on the microarray slide was probed with anti-V5-Alexa 647 antibody. (B) HEK293T cells were transfected with either FLAG-tagged GSK-3β, β-catenin, ZNRF3, RNF43, FZD6, LRP6, DVL2, or empty vector (EV) and HA-tagged LMBR1L. Lysates were subsequently immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with antibodies against HA or FLAG. (C) Immunoblot analysis of β-catenin, phospho-β-catenin, AXIN1, DVL2, GSK-3α/β, phospho-GSK-3β, CK1, β-TrCP, c-Myc, p53, p21, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and GAPDH in TCLs of pooled CD8+ T cells from Lmbr1l−/− or wild-type littermates. Data are representative of three-to-five independent experiments.

Article Snippet: Protein array The ProtoArray Human Protein Microarray V5.1 (Invitrogen) was used to identify human Lmbr1l–interacting proteins according to manufacturer`s instructions.

Techniques: Microarray, Binding Assay, Construct, Expressing, Transfection, Recombinant, Purification, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A) Manhattan plot. −Log10 P-values plotted vs. the chromosomal positions of mutations identified in the affected pedigree. (insets) Representative flow cytometric plot of B220+ and CD3+ peripheral blood lymphocytes in wild-type (WT) and strawberry mice. (B) LMBR1L topology. The schematic shows the location of the Lmbr1l point mutation, which results in substitution of cysteine 212 for a premature stop codon (C212*) in the LMBR1L protein. (C-J, M, N) Frequency and surface marker expression of T (C-F), B (H-J), NK (M), and NK1.1+ T (N) cells in the peripheral blood from 12-week-old Lmbr1l−/− or Cers5−/− mice generated by the CRISPR/Cas9 system. (K) T cell-dependent β-gal-specific antibodies 14 days after immunization of 12-week-old Lmbr1l−/− or Cers5−/− mice with a recombinant SFV vector encoding the model antigen, β-gal (rSFV-βGal). Data presented as absorbance at 450 nm. (L) T cell-independent NP-specific antibodies 6 days after immunization of 13-week-old Lmbr1l−/− or Cers5−/− mice with NP-Ficoll. Data presented as absorbance at 450 nm. (O) Quantitative analysis of the β-gal-specific cytotoxic T cell killing response in Lmbr1l−/− mice that were immunized with rSFV-βGal. An equal mixture of ICPMYARV peptide (β-gal-specific MHC I epitope for mice with H-2d haplotype) pulsed CFSEhi and unpulsed CFSElo splenocytes were adoptively transferred to immunized mice by retro-orbital injection. Mice were bled 48 h following adoptive transfer and killing of CFSE-labeled target cells was analyzed by flow cytometry. (P) Lmbr1l−/− mice generate reduced antigen-specific CD8+ T cell responses to aluminum hydroxide precipitated ovalbumin (OVA/alum). Lmbr1l−/− and wild-type littermates were immunized with OVA/alum at day 0. Total and memory Kb/SIINFEKL tetramer-positive CD8+ T cells were analyzed at day 14 by flow cytometry using CD44 and CD62L surface markers. (Q) NK cell cytotoxicity against MHC class I-deficient (B2m−/−) target cells in Lmbr1l−/− mice. An equal mixture of CellTrace Violet-labeled C57BL/6J (Violetlo) and B2m−/− (Violethi) cells were transferred into recipient mice and NK cell cytotoxicity toward target cells was analyzed by flow cytometry 48 h after injection. (R) Viral DNA copies in livers from Lmbr1l−/− mice 5 days after infection with 1.5 × 105 pfu MCMV Smith strain. Each symbol represents an individual mouse (C-R). P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons (C-O, Q, R) or Student’s t-test (P). Data are representative of two independent experiments (C-J, M, N) or one experiment (K, L, O-R) with 5–24 mice per genotype. Error bars indicate S.D. * P < 0.05; *** P < 0.001.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Mutagenesis, Marker, Expressing, Generated, CRISPR, Recombinant, Plasmid Preparation, Injection, Adoptive Transfer Assay, Labeling, Flow Cytometry, Infection

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A-D) Repopulation of lymphocytes in spleen (A, B), thymus (C), and bone marrow (D) 12 weeks after reconstitution of irradiated wild-type (C57BL/6J; CD45.1) and strawberry (CD45.2) recipients with strawberry (CD45.2) or wild-type (C57BL/6J; CD45.1) bone marrow, or Rag2−/− recipients with a 1:1 mixture of Lmbr1lst/st (CD45.2) and wild-type (C57BL/6J; CD45.1) bone marrow. Representative flow cytometric scatter plots of B and T cells (A), NK cells (B), thymocytes (C), and bone marrow B cells. MR: mature recirculating B cells, Trans.: transitional B cells, Imm.: immature B cells. Numbers adjacent to outlined areas or in quadrants (A-D) indicate percent cells in each. (A, B, E-G) Reconstitution of B (A, E), T (A, F), and NK (B, G) cells in the spleen of recipients with donor-derived cells 12 weeks after engraftment. (C, D, H-K) Repopulation of donor-derived T cell subsets in thymus (C, H, I) and B cell subsets in bone marrow (D, J, K) in recipients rescued from lethal irradiation. (L, M) The frequencies (L) and total numbers (M) of stem and progenitor cell subsets per femur in the LSK+ and LK+ compartments in the bone marrow of Lmbr1l−/− and wild-type littermates as determined by flow cytometry. Each symbol represents an individual mouse (E-M). P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons (E-K) or Student’s t-test (L, M). Data are representative of two independent experiments with 6–7 mice per genotype. Error bars indicate S.D. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Irradiation, Derivative Assay, Flow Cytometry

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A) LMBR1L-deficient peripheral T cells are activated. Flow cytometric analysis of CD44 expression on T cells in the thymi and spleens of 12-week-old Lmbr1l−/− and wild-type littermates. (B) Immunoblot analysis of TCF1/7, LEF1, Akt, phospho-Akt, S6, phospho-S6, phospho-p70S6K, phospho-p44/p42 MAPK, and GAPDH in total cell lysates (TCLs) of pooled CD8+ T cells from Lmbr1l−/− or wild-type littermates. (C) Annexin V staining of CD4+ or CD8+ T cells in peripheral blood obtained from 14-week-old wild-type or strawberry mice. (D) IL-7Rα expression on CD3+ T cells in peripheral blood obtained from 12-week-old Lmbr1l−/− and wild-type littermates. (E-G) Impaired antigen-specific expansion of LMBR1L-deficient T cells. A 1:1 mixture of CellTrace Violet-labeled Lmbr1l−/− (CD45.2) and Far Red-stained wild-type OT-I T cells (CD45.2) was adoptively transferred into wild-type hosts (C57BL/6J; CD45.1). Representative flow cytometric scatter plots (E) and histograms (F), and quantification of total numbers (G) of CellTrace Violet- or Far Red-positive wild-type or Lmbr1l−/− OT-I T cells harvested from the spleens of wild-type (C57BL/6J; CD45.1) hosts, 48 or 72 h after immunization with soluble OVA or sterile PBS (vehicle) as a control. (H-M) Impaired homeostatic expansion of LMBRlL-deficient T cells. An equal mixture of CellTrace Violet-labeled or CellTrace Far Red-stained pan T cells isolated from the spleen (H-J) or mature single-positive thymocytes (K-M) from Lmbr1l−/− or wild-type littermates were adoptively transferred into sublethally irradiated (8.5 Gy) wild-type hosts (C57BL/6J; CD45.1). Representative flow cytometric scatter plots (H, K) and histograms (I, L), and quantification of total numbers (J, M) of CellTrace Violet- or CellTrace Far Red-positive cells harvested from the spleens of sublethally irradiated or unirradiated wild-type hosts, 4 or 7 days after transfer. Numbers adjacent to outlined areas indicate percent cells in each ± SD. Each symbol represents an individual mouse (A, C, D, G, J, M). P-values were determined by Student’s t-test (A, C, D) or one-way ANOVA with Dunnett’s multiple comparisons (G, J, M). Data are representative of two independent experiments with 4–29 mice per genotype or group. Error bars indicate S.D. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Expressing, Western Blot, Staining, Labeling, Isolation, Irradiation

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: LMBR1L-interacting proteins identified by co-immunoprecipitation (co-IP) combined with mass spectrometry (MS) analysis that were increased more than 50-fold, or Wnt components exclusively present in the LMBR1L co-IP relative to empty vector control.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Mass Spectrometry, Plasmid Preparation

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A, B) LMBR1L physically interacts with components of the Wnt signaling pathway. (A) Human protein microarray revealed binding between LMBRlL and GSK-3β proteins. A construct expressing both N-terminus FLAG-tagged and C-terminus V5-tagged human LMBR1L was transfected into HEK293T cells and the recombinant protein was purified using anti-FLAG M2 agarose beads. Binding between recombinant human LMBR1L and purified human proteins printed in duplicate on the microarray slide was probed with anti-V5-Alexa 647 antibody. (B) HEK293T cells were transfected with either FLAG-tagged GSK-3β, β-catenin, ZNRF3, RNF43, FZD6, LRP6, DVL2, or empty vector (EV) and HA-tagged LMBR1L. Lysates were subsequently immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with antibodies against HA or FLAG. (C) Immunoblot analysis of β-catenin, phospho-β-catenin, AXIN1, DVL2, GSK-3α/β, phospho-GSK-3β, CK1, β-TrCP, c-Myc, p53, p21, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and GAPDH in TCLs of pooled CD8+ T cells from Lmbr1l−/− or wild-type littermates. Data are representative of three-to-five independent experiments.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Microarray, Binding Assay, Construct, Expressing, Transfection, Recombinant, Purification, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A) Immunoblots of the indicated proteins in membrane and TCLs of pooled CD8+ T cells isolated from the spleens of 12-week-old Lmbr1l−/− or wild-type littermates. The upper band of FZD6 or LRP6 (red arrowhead) is the mature form; the lower band (blue arrowhead) is the ER form of FZD6 or LRP6 (also applies to B, C, E, F). Expression of GRP94 or BiP was determined with a KDEL antibody. GAPDH was used as loading control. *, an unknown KDEL-positive protein whose expression is unchanged. (B) HEK293T cells were transfected with FLAG-tagged FZD6 and either HA-tagged LMBR1L, UBAC2, GP78, or empty vector. TCLs were immunoprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, HA, and Ubiquitin (UB). GAPDH was used as a loading control. (C) HEK293T cells were transfected with FLAG-tagged LRP6 and HA-tagged LMBR1L, UBAC2, or empty vector. TCLs were immunoblotted using the indicated antibodies. (D) ER or plasma membrane proteins were isolated from LMBR1L-FLAG knock-in (KI) or parental HEK293T cells (WT). Endogenous LMBR1L expression was then analyzed by immunoblotting using a FLAG antibody. Expression of calnexin, E-cadherin, or α-tubulin were used as loading controls for ER, plasma membrane, or cytosol, respectively. (E) Immunoblots of indicated proteins in TCLs of pooled CD8+ T cells isolated from the spleens of 6-week-old Gp78−/− or wild-type mice. (F) Constructs encoding FLAG-tagged LRP6 and HA-tagged LMBR1L were transfected into Gp78−/− or parental HEK293T cells. TCLs were immunoblotted using the indicated antibodies. (G) HEK293T cells were transfected with FLAG-tagged β-catenin and either HA-tagged LMBR1L, UBAC2, GP78, or empty vector. TCLs were immunoprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, HA, and Ubiquitin (UB). GAPDH was used as a loading control. Data are representative of two-to-five independent experiments.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Western Blot, Isolation, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Knock-In, Construct

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A) HEK293T cells were transfected with FLAG-tagged GSK-3β and either HA-tagged LMBR1L or empty vector. TCLs were immuno-precipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against p-GSK-3β, FLAG, and HA. GAPDH was used as a loading control. (B) HEK293T cells were transfected with FLAG-tagged GSK-3β and either HA-tagged LMBR1L or empty vector. The cells were treated with cyclohexamide (CHX) 14 h after transfection and harvested at various times post-treatment. TCLs were immunoblotted with the indicated antibodies. Two primary antibodies (anti-HA and GAPDH) were co-incubated to visualize LMBR1L (red arrowhead) and GAPDH (blue arrowhead, a loading control) on one membrane. Data are representative of three independent experiments.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Transfection, Plasmid Preparation, Incubation

Journal: Science (New York, N.Y.)

Article Title: LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

doi: 10.1126/science.aau0812

Figure Lengend Snippet: (A-C) Growth curve (A) and Annexin V/PI staining (B) of Lmbr1l−/−, Ctnnb1−/−, and Lmbr1l−/−;Ctnnb1−/− EL4 cells generated by the CRISPR/Cas9 system (n = 3–5 clones/genotype), and parental wild-type (WT) EL4 cells. Numbers adjacent to outlined areas (B) indicate percent cells in each. (C) Quantification of the percentage of viable, apoptotic, and necrotic Lmbr1l−/−, Ctnnb1−/−, Lmbr1l−/−;Ctnnb1−/−, or parental WT EL4 cells. Each symbol represents an individual cell clone. P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of three independent experiments. Error bars indicate S.D. * P < 0.05; ** P < 0.01.

Article Snippet: Purified recombinant Flag -human LMBR1L-V5 was used to probe a Human V5.1 ProtoArray (Invitrogen) at a final concentration of 50 μg/ml.

Techniques: Staining, Generated, CRISPR, Clone Assay

Journal: iScience

Article Title: SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

doi: 10.1016/j.isci.2022.105183

Figure Lengend Snippet: WNT5A may link SEL1L-HRD1 ERAD to hepatic proliferation (A and B) Volcano plot (A) and tabular form (B) of proteomics data obtained via TMT-LC/MS analysis of hepatic ER isolated from 2- to 3-months-old mice (n = 3 per group). Lines mark p = value 0.05 (horizontal) or 2-fold changes (vertical). (C–F) Western blot analysis of ER and non-ER fractions from livers of 2-month-old mice (n = 3 per group) (C, black and red arrows mark WNT5A mobility shift), hepatocellular carcinoma and normal liver tissue from the DEN-HFD liver cancer model (n = 2 per group, 2 independent repeats) (D), livers of 10-week-old mice 48-h post partial hepatectomy (n = 3 per group) (E), livers from chronic CCL4 treatment (n = 2 per group) (F). (G) Western blot analysis normal liver and tumor tissues from the year-long-HFD experiment (n = 3 per group). (H and I) Hepatic cDNA microarray analyses of 9-week-old Sel1L Alb livers compared to Sel1L f/f livers: (H) KEGG pathway for Gene Set Enrichment Analysis (GSEA) from differentially expressed hepatic genes with p < 0.05 and fold change >1.29, and (I) Heatmap of proliferation-associated genes as a logarithm of fold change in Sel1L Alb and Hrd1 Alb livers as compared to Sel1L f/f and Hrd1 f/f livers, respectively (n = 3 per group). (J) Western blot analysis of HepG2 and Huh7 cells treated with 500 ng/mL recombinant WNT5A protein for 30 h (2 independent repeats). (K) Western blot analysis of Sel1L f/f and Sel1L Alb livers 2 weeks after i.v. injection of AAV8 expressing shRNA targeting Wnt5A or luciferase control (n = 2–3 per group). Quantitation shown below each blot. Calnexin, ER fraction control; HSP90, loading and non-ER controls. Values, mean ± SEM; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by Student’s t test.

Article Snippet: The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Western Blot, Mobility Shift, Microarray, Recombinant, Injection, Expressing, shRNA, Luciferase, Quantitation Assay

Journal: iScience

Article Title: SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

doi: 10.1016/j.isci.2022.105183

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors).

Techniques: Western Blot, Plasmid Preparation, Microarray, Recombinant, Purification, Mutagenesis, Software, Expressing