Journal:

Article Title: Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules

doi: 10.1093/emboj/21.7.1650

Figure Lengend Snippet: Fig. 4. The CD1d–class II association exists in MIICs and on the cell surface. (A) C1R.CD1d cells were disrupted by a ball-bearing homogenizer and fractionated using a Percoll density gradient. Aliquots of each fraction were analyzed by SDS–PAGE followed by immunoblotting with D5, R.DRAB (rabbit anti-DR), DM323 (rabbit anti-DM) and MaP.ERp57 (anti-ERp57 mAb). Fraction 1 corresponds to the top of the gradient. (B) CD1d–class II complexes were immunoprecipitated from Brij 98-solubilized fractions using L243 and analyzed by SDS–PAGE followed by immunoblotting with D5 or R.DRAB. (C) C1R.CD1d cells were radiolabeled for 15 min and chased for 30 min (lanes 1 and 3) or 5 h (lanes 2 and 4). Cell surface proteins were biotinylated with sulfo-NHS-SS-biotin (see Materials and methods) prior to extraction in 1% Brij 98. The extracts were immunoprecipitated with either 51.1.3 (lanes 1 and 2) or L243 (lanes 3 and 4). Precipitated proteins were dissociated from the beads in non-reducing elution buffer containing 1% SDS and the eluates re-precipitated with streptavidin–agarose prior to separation by 12% SDS–PAGE. The bands corresponding to CD1d, and DRα and β chains, are marked on the right of the gel.

Article Snippet: To detect CD1d on SDS–PAGE, D5 mAb, purified using protein A–Sepharose, was conjugated with sulfo-NHS-SS-biotin (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions and used for western blotting with HRP-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and enhanced chemiluminescence (Pierce).

Techniques: SDS Page, Western Blot, Immunoprecipitation

Journal:

Article Title: Control of her1 expression during zebrafish somitogenesis by a Delta -dependent oscillator and an independent wave-front activity

doi:

Figure Lengend Snippet: aei/DeltaD is required for both neurogenesis and patterning of aei/DeltaD expression in the PSM. (A–C) Examination of Islet-1-expressing neurons (black/blue) in the trunk of aei embryos revealed a neuronal hyperplasia. Using myosin (brown) as a reference for somite position, Islet-1-expressing neurons anterior to the posterior border of somite 5 were counted in wild-type (A) and aei embryos (B,C). Wild-type embryos (n = 20) averaged 33 Islet-1-expressing neurons in this region, whereas aeiAG49 (n = 19) and aeiAR33 (n = 20) averaged 58 and 61 such neurons, respectively. All embryos are at about the 13-somite stage. Photos show dorsal views of the trunk region. (D–I) Expression of aei/DeltaD is mispatterned in each of the fss-type mutants. Note, in fss embryos (F), 10%–15% show some evidence of aei/DeltaD stripe formation. Most embryos, however, exhibit expression patterns as seen in aei/DeltaD and the other fss-type mutants. All embryos are at about the 15-somite stage. Photos show dorsal views of the tailbud and posterior trunk. In all panels, anterior is to the left and posterior to the right.

Article Snippet: For antibody stainings, embryos were fixed in 4% PFA, blocked, incubated with the α-Islet-1 antibody (39.4D5 monoclonal, 1:500 dilution; Developmental Studies Hybridoma Bank), washed, incubated with 2% biotinylated anti-mouse (Vector), washed, and stained according to standard procedures.

Techniques: Expressing