Journal:

Article Title: Functional cleavage of the common cytokine receptor ? chain (? c ) by calpain

doi:

Figure Lengend Snippet: Cytokine receptor subunits with PEST sequences in their cytoplasmic domains

Article Snippet: Immunoprecipitations were performed with immobilized anti-chicken IgY (Promega) and either chicken anti-human γ c antibody (Promega) or chicken IgY (H+Y) as a control antibody (Promega).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

doi: 10.1093/nar/gkn413

Figure Lengend Snippet: Detection of IBtkα, IBtkβ and IBtkγ endogenous proteins. ( A ) IBtkα (150.53 kDa) and IBtkβ (133.87 kDa) were detected by western blot analysis of DeFew cell extracts (150 μg) by using the anti 31B IgY antibody (10 μg/ml) raised in chickens by using the 31B peptide, which was shared by IBtkα and IBtkβ isoforms. The antibody specificity was assessed by using either the competitor 31B peptide (60 nmoles/ml, lane 2), or a 31B scrambled peptide (60 nmoles/ml, lane 1) in competition experiments. Input proteins were equalized by detecting the endogeneous γ-tubulin. ( B ) IBtkα (150.53 kDa) and IBtkγ (26.31 kDa) were detected by western blot analysis of DeFew cell extracts (100 μg) by using an anti-GST-IBtkγ serum (1:500). The antibody specificities were assessed by using GST (60 nmoles/ml, lane 2), or GST-IBtkγ (60 nmoles/ml, lane 3) proteins in competition experiments. The preimmune serum was unreactive (lane 4). Input proteins were equalized by detecting the endogeneous γ-tubulin. ( C ) Expression of IBtk isoforms. Protein extracts from 293T, HeLa and lymphoblastoid cells (DeFew, Jurkat, MC3, NB4) (50 μg to detect IBtkα and IBtkβ; 100 μg to detect IBtkγ) were tested in western blots by using the indicated antibodies. Input proteins were equalized by detecting the endogeneous γ-tubulin. ( D ) Expression of IBtkα and Ibtkγ in mouse tissues. Western blot analysis was performed by testing 50 μg of protein extracts from mouse tissues. To detect IBtkγ, we used anti GST-IBtkγ serum as primary antibody. IBtkα was detected by the anti-31B IgY. Consistent with the gene organization of mouse Ibtk , the mouse IBtkβ isoform was not detected. ECL Plus was performed to detect human IBtkγ.

Article Snippet: IBtkα and IBtkβ were detected by using the chicken 31B IgY antibody (10 μg/ml) followed by incubation with a anti-chicken-HRP (Gallus Immunotech Inc.).

Techniques: Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

doi: 10.1093/nar/gkn413

Figure Lengend Snippet: Physical interaction of IBtk with Btk, Itk, Akt and PLCγ1. The in vivo presence of protein complexes of the distinct IBtk isoforms with Btk, Itk, Akt and PLCγ 1 was assessed by sequential immunoprecipitation and western blot analysis. Primary PBMC were subjected to immune precipitation with anti-Btk, anti-Itk, anti-Akt, anti-PLCγ 1 and mouse IgG 1 control antibody. To detect IBtkα, IBtkβ, IBtkγ, immunoprecipitates were analyzed by western blotting (WB) by using the anti-31B IgY antibody (upper panel) to detect IBtkα and IBtkβ, and the anti-GST-IBtkγ antibody to detect IBtkγ (middle panel). The lower panel shows the expression of the tested proteins as detected by western blotting with the anti-Btk, anti-Itk, anti-Akt and anti- PLCγ 1 -specific antibodies.

Article Snippet: IBtkα and IBtkβ were detected by using the chicken 31B IgY antibody (10 μg/ml) followed by incubation with a anti-chicken-HRP (Gallus Immunotech Inc.).

Techniques: In Vivo, Immunoprecipitation, Western Blot, Control, Expressing

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 1. The amino acid residues of hCRM1 required for Rex ac- tivity. (A) The ability of CRM1s to support Rex activity. HeLa cells were transfected with the indicated plasmids. After cell lysis, the amount of CAT and the activity of -Gal were measured and CAT/ -Gal ratios were calculated. The ratio for the control sample without pSRTAgRexM64 and pSRCRM1s was arbitrarily set at 1. The amount of CAT and the -Gal activity in control samples were over 300 pg and 3.0 103 U, respectively. Error bars represent standard deviations. (B) Restoration of Rex activity by overexpression of CRM1s in HeLa cells. The experimental procedure was the same as that described for panel A. (C) Effect of overexpressing CRM1s on Rex activity in REF52 cells. REF52 cells were transfected with the indicated plasmids. At 24 h of posttransfection, CAT/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbi- trarily set at 1. (D) Western blot analysis of various CRM1s. A fraction of each sample used in the experiments described for panel C was subjected to Western blot analysis using the anti-hCRM1 antibody to examine CRM1 protein synthesis. This antibody was raised with the peptide, which represents the carboxy-terminal region of hCRM1 and has a sequence different from that of rCRM1, so it does not recognize endogenous rCRM1. (E) Effect of CRM1s on Rex-mediated Gag expression from HTLV-1 molecular clone. At 48 h posttransfection, Gag/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbitrarily set at 1. (F) A schematic representation of hCRM1 functional domains. The amino acid sequence of hCRM1 is shown. Experiments characterizing the RanGTP binding domain (bro- ken line) (33), LMB binding residue (arrowhead) (24), Rev-interacting amino acids (asterisks) (2), and the domain binding to NES (under- line) (33) have been previously reported. The residues 411, 414, 474, and 481 in hCRM1 are indicated in bold characters, and the corre- sponding residues of rCRM1 are indicated under the hCRM1 se- quence in single-letter amino acid code.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Activity Assay, Transfection, Lysis, Control, Over Expression, Western Blot, Sequencing, Expressing, Functional Assay, Binding Assay, Residue

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 2. In vivo interaction of Rex with CRM1 mutants in which one amino acid is replaced. REF52 cells were transfected with the plasmid expressing GAL-CRM1s in combination with pRexVP, pG5BCAT, and pCDM-gal. The cells were harvested and subjected to CAT and -Gal assays, and CAT/-Gal ratios were calculated. The ratio for the control sample, which detected the interaction between GAL-hCRM1 and Rex-VP, was arbitrarily set at 1. The amount of CAT and -Gal activity in control samples were over 400 pg and 3.0 103 U, respec- tively. GAL4 nonfusion protein, expressing only a GAL4 region, was used as a negative control. A fraction of each sample was subjected to Western blot analysis using the anti-GAL4 monoclonal antibody to examine GAL-CRM1 expression.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Control, Activity Assay, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 6. Binding characterization of two amino acid-substituted CRM1s. (A) In vivo interaction of CRM1s with Rex. REF52 cells were treated as described for Fig. 2 except for the plasmids that expressed two amino acid-substituted CRM1s as a GAL4 fusion protein. A portion of each sample was subjected to Western blot analysis to confirm GAL-CRM1 expression. (B) In vitro binding of h411/414 to RanBP3. As shown in Fig. 5A, in vitro-translated CRM1s were incubated with GST or GST-RanBP3 immobilized on glutathione-Sepharose 4B.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Binding Assay, In Vivo, Western Blot, Expressing, In Vitro, Incubation

Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 7. Blocking Src activity reduces proteinuria in msuPAR2-Tg mice. (A) Glomerular Src activity was determined by immunofluorescent staining of the kidney cryosections with p-Src monoclonal antibody (green). Podocin was used as a podocyte marker (red). Shown are the representative glomeruli with p-Src immunofluorescent staining. As indicated by the p-Src staining intensity, glomerular Src phosphorylation was readily observed in the kidney sections from msuPAR2-Tg, but not from other investigated mice. Scale bars: 20 μm. (B) Treatment with Src inhibitors lowered proteinuria in msuPAR2-Tg mice. Both PP1 and Src inhibitor 1 (5 mg/kg) were administered into the randomly grouped msuPAR2-Tg mice intraperitoneally, with vehicle controls receiving the same amount of DMSO. n = 8 Src inhibitor group; n = 7 vehicle control group. At 72 hours after treatment, proteinuria was significantly reduced by Src inhibitor treatment. Two-way ANOVA; data were log-transformed to normal distribution. **P < 0.01.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Blocking Assay, Activity Assay, Staining, Marker, Phospho-proteomics, Control, Transformation Assay

Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 8. Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immu- nostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors (n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was local- ized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE (n = 2), MPGN (n = 2), and MCD (n = 4). Scale bar: 20 μm. NT, normal kidney tissue.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Staining, Marker, Phospho-proteomics