rabbit anti-mouse cxcl12  (Torrey Pines Biolabs)

Journal: Nature Communications

Article Title: Regulation of T cell afferent lymphatic migration by targeting LTβR-mediated non-classical NFκB signaling

doi: 10.1038/s41467-018-05412-0

Figure Lengend Snippet: Agonistic anti-LTβR mAb induces inflammatory and homeostatic chemokines and cell adhesion molecules in LEC. a qRT-PCR of indicated genes induced by agonistic anti-LTβR mAb or isotype rat IgG crosslinked with or without mouse anti-rat IgG1 in LEC at the indicated times. Treated as in Fig. b Flow cytometry analysis of VCAM-1 expression on LEC after treatment with anti-LTβR mAb plus crosslinking, with or without 25 μM NFκB inhibitor BAY11-7085 or 50 μM NIKi for 3 h. c LTβR-induced CXCL12 and VCAM-1 expression on SVEC4-10 treated with anti-LTβR mAb plus crosslinking, with or without NIKi (50 μM) for 3 h. Magnification ×60; scale bar 5 μm. d Secretion of CCL2 and CCL21 by LEC stimulated with anti-LTβR mAb plus crosslinking, with or without BAY11-7085 (25 μM) or NIKi (50 μM) measured at the indicated times. * p < 0.05 by one-way ANOVA ( a , d ) or by Student’s t -test ( c )

Article Snippet: Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), and treated with 4% donkey serum for 30 min then incubated with rabbit-anti-mouse CXCL12 (eBioscience), goat-anti-mouse CCL21 (Clone AF457; R&D System), anti-NIK mouse monoclonal antibody (Clone A-12; Santa Cruz Biotechnology), or rabbit-anti-mouse RelA (Cell Signaling) or RelB (Thermo Fisher Scientific) for nuclear translocation staining overnight at 4 °C.

Techniques: Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Regulation of T cell afferent lymphatic migration by targeting LTβR-mediated non-classical NFκB signaling

doi: 10.1038/s41467-018-05412-0

Figure Lengend Snippet: Regulation of gene profiles by the LTβR NFκB-blocking peptides. a qRT-PCR of VCAM-1 and chemokines in LEC stimulated with anti-LTβR mAb (2 μg/mL) plus CL with mouse anti-rat IgG1 (2 μg/mL) for the indicated times. b ELISA for CCL2 and CCL21 in the LEC supernatants after 4 or 16 h stimulation as in a , respectively. c CXCL12 and VCAM-1 expression in LEC after 3 h stimulation as in a . Magnification ×40; scale bar 20 μm. d C57BL/6 mice injected with 5 nmol/ear control peptide (CP), nciLT or ciLT; after 3 h ears were fixed for whole-mount ear staining. Magnification ×20; scale bar 50 μm. e qRT-PCR of VCAM-1 in LEC stimulated with TNFα (20 ng/mL) for 1 h. f , g Whole-mount ear staining of NIK and CCL21 in Prox1-Cre-ER T2+/− LTβR fl/fl mice 10 days after tamoxifen treatment (0.125 mg/g i.p. for 5 consecutive days). BV blood vessel. Magnification ×60; scale bar 10 μm. * p < 0.05 by one-way ANOVA ( a – e ) or by Student’s t- test ( f , g )

Article Snippet: Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), and treated with 4% donkey serum for 30 min then incubated with rabbit-anti-mouse CXCL12 (eBioscience), goat-anti-mouse CCL21 (Clone AF457; R&D System), anti-NIK mouse monoclonal antibody (Clone A-12; Santa Cruz Biotechnology), or rabbit-anti-mouse RelA (Cell Signaling) or RelB (Thermo Fisher Scientific) for nuclear translocation staining overnight at 4 °C.

Techniques: Blocking Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Injection, Control, Staining

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) The structures of the bait plasmids. (i) CXCL12 was fused with a signal peptide (SP) at the N-terminus and with a transmembrane peptide (TMP) at C-terminus, followed by C ub and GAL4 in tandem. (ii) WBP1 was fused with C ub and GAL4 as a control. Bait genes were cloned into the plasmid pGAD-T7. ( B ) Structures of prey plasmids. (iii) The prey fusion was composed of a SP, CXCR and N ub G in tandem. (iv) OST1 was fused with N ub G as a control prey. Prey genes were cloned into the plasmid pGBK-T7. ( C ) Principles of detecting the interaction. (v) The bait protein is expressed as a transmembrane protein, in which the CXCL12 domain is directed into the lumen (and the periplasm, if mature forms are located on plasmalemma) and linked with the C ub -GAL4 cassette in the cytoplasm by the TMP. CXCRs-N ub G is co-expressed on the plasma membrane. The interaction of CXCL12 and CXCRs unites C ub and N ub G and leads to the reconstitution of the split ubiquitin protein, which is recognized and cleaved by UBP to release GAL4. The liberated GAL4 protein is transported into the nucleus by the SV40 nuclear localization signal at its N-terminus, and transcription of reporter genes lacZ , HIS3 and ADE2 results in blue clones on a SD/His − Ade − plate in the presence of α -X-Gal. (vi) Coexpression of WBP1-C ub -GAL4 and OST1-N ub G was used as the positive interacting proteins control. The two genes are known interacting partners of membrane proteins of yeast. (vii) Coexpression of SP-CXCL12-TMP-C ub -GAL4 and OST1-N ub G , two unrelated genes, was used as a negative control.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Clone Assay, Plasmid Preparation, Negative Control

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) Structure of bait plasmids. The pGAD-T7 vector was used as the backbone of the bait plasmids, in which bait genes were promoted by the ADH1 promotor. Three different signal peptides, (i) SP CXCL12 , (ii) SP WBP1 , or (iii) SP MFAL1 were fused at the N-terminal end of CXCL12, and the transmembrane peptide of TMP WBP1 and EGFP were fused to the C-terminus of CXCL12 in tandem. (iv) WBP1-EGFP was used as the positive control. ( B ) Structures of prey plasmids. The pGBK-T7 vector was used as the backbone of the prey plasmids, in which preys were promoted by the ADH1 promoter. (v) CXCR4 with the native N-terminus or CXCR4 fused with different signal peptides, (vi) SP WBP1 , (vii) SP OST1 , or (viii) SP MFAL1 were tagged with EGFP at the C-terminus. (ix) OST1-EGFP was used as the control. ( C,D ) LSCM images of EGFP. Baits (i’-iv’) and preys (v’-ix’) were expressed by GoldY2H and Y187, respectively, and observed with LSCM. Images of EGFP and membrane-specific fluorescent dye DiI and transmission light were captured and merged. The bars indicate 10 micrometers. The arrows direct the membrane localization of baits and preys, and the asterisks indicate the unexpected intracellular localizations.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Plasmid Preparation, Positive Control, Transmission Assay

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) Self-activation detection. GoldY2H cells expressing bait protein with SP CXCL12 , SP WBP1 and SP MFAL1 signal peptides were detected to self-activate. Five microliter diluted suspensions were dropped onto SD/Trp − Ade − His − plates with α -X-gal and 3-AT, and grown for 72 h. ( B ) Cells growth assay. GoldY2H cells expressing the SP CXCL12 -CXCL12-TMP-C ub -GAL4 plasmid was hybridized with Y187 cells expressing each SP OST1 -CXCR-N ub G fusion protein, and diploid cells were dropped onto SD/Leu − Trp − Ade − His − plates containing α -X-gal and 15 mM 3-AT. The growth of cells was observed after 72 h cultivation. SP CXCL12 -CXCL12-TMP-C ub -GAL4 × OST1-N ub G were used as a negative control, and WBP1-C ub -GAL4 × OST1-N ub G were used as a positive control. ( C ) Growth assay of cells expressing SP CXCL12 -CXCL12-TMP-C ub -GAL4 and CXCR-N ub I. ( D ) Quantitative β-Gal assay of diploid cells expressing SP-CXCL12-TMP-Cub-GAL4 and each of the SP OST1 -CXCR-N ub G proteins. The experiment was repeated three times independently and every sample was tested twice in each experiment ( n = 2). ( E ) Western blot analysis of cleaved GAL4. Diploid cells expressing SP CXCL12 -CXCL12-TMP-C ub -GAL4 and each of the CXCR-N ub G proteins were lysed and the GAL4 level detected by western blot analysis. The molecular weight (MW) of SP CXCL12 -CXCL12-TMP-C ub -GAL4 is 59 kDa, and the MW of cleaved GAL4 is 31 kDa. The experiment was performed three times independently. The asterisk denotes a non-specific band.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Activation Assay, Expressing, Growth Assay, Plasmid Preparation, Negative Control, Positive Control, Western Blot, Molecular Weight

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) Structures of chimeric receptors of CXCR5 and CXCR3. Four extracellular fragments of CXCR5 were exchanged with counterparts of CXCR3 to form eight chimeric receptors. ( B ) Cell growth detection. Diploid cells expressing SP CXCL12 -CXCL12-TMP-C ub -GAL4 and CXCR5-N ub G , CXCR3-N ub G , or N ub G tagged chimeric receptors were detected by the growth ability on selection plate (SD-Leu − Trp − Ade − His − ) with 15 mM 3AT + α-X-gal (left column). Diploid cells harboring the bait and N ub I tagged preys were detected (right column). Positive and negative control cells were the same as . ( C ) Expression detection by western blot assay. The chimeric preys labeled with EGFP were detected by western blot. ( D ) Quantitative β-Gal assay of diploid cells expressing SP CXCL12 -CXCL12-TMP-C ub -GAL4 and N ub G tagged chimeric receptors. The experiment was repeated three times independently and every sample was tested twice in each experiment ( n = 2). * and *** indicate significance p < 0.05 and p < 0.001 of a t-test between chimeric receptors and wild-type counterparts.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Expressing, Selection, Negative Control, Western Blot, Labeling

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) The interaction between CXCL12 and the N-terminus of CXCR5 or CXCR3 was detected by a traditional Y2H, in which CXCL12 was fused with the activation domain of GAL4 (AD) as the bait, and CXCR5 NT or CXCR3 NT was fused with the binding domain (BD) of GAL4 as the preys. Only yeast expressing AD-CXCL12 and BD-CXCR5 NT could survive the selective medium, indicating the interaction between prey and bait. ( B ) CoIP of CXCL12 and CXCR5 NT . CXCL12 and CXCR5 NT -Fc or CXCR3 NT -Fc were co-transfected into the 293T cell line. Fc-tagged fusion proteins were precipitated by protein G beads, and the precipitates were separated by SDS-PAGE and blotted with rabbit anti-mouse CXCL12 IgG. The result shows that CXCL12 was precipitated by CXCR5 NT -Fc, but not by CXCR3 NT -Fc or Fc only, indicating an interaction between CXCR5 NT and CXCL12.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Activation Assay, Binding Assay, Expressing, Transfection, SDS Page

Journal: Scientific Reports

Article Title: Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

doi: 10.1038/srep35631

Figure Lengend Snippet: ( A ) CXCL7 and CXCL9 were detected the PPIs with receptors of CXCR family (CXCRn-N ub G) using growth assay, and the result showed different PPIs intensity with each receptors, in which obvious PPIs were detected between CXCL7 and CXCR2 and between CXCL9 and CXCR3. ( B ) Interleukin ligand/receptor pairs, IL1A/IL1R1 and IL6/IL6RA, were detected the interactions. CXCL12 and CXCR4 was used as negative controls of bait and prey respectively, and Ost1-N ub I was used as positive prey control. Through growth assays, the expected interactions of IL1A/IL1R1 and IL6/IL6RA were detected correctly, meanwhile, no crosstalk was detected between unmatched ligand/receptor pairs.

Article Snippet: GAL4 was detected by a rabbit anti-GAL4 IgG (abcam, ab1396), and CXCL12 by a rabbit anti-mouse CXCL12 IgG (abcam, 9797).

Techniques: Growth Assay