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This study was performed on cryosections, cell culture monolayers, blood cell smears and cytospins fixed for no longer than 15 min in either acetone or alcohol.
P. pastoris culture supernatants were denatured by boiling for 5 min in either reducing or non-reducing SDS sample buffer (0.0625 M Tris HCl, pH 6.8, 2.3% (w/v) SDS, 10% (w/v) glycerol and 0.01% Bromophenol blue.
Briefly, viruses were incubated at 45°C for 0, 5, 10, 20 or 40 min in either PBS (without Ca2+ and Mg2+) or growth medium containing 2% FBS.
Scf-evoked phosphorylation of these residues at 5 min in either LSK or LK cells was primarily UO126-, not rapamycin-sensitive, and therefore MEK, rather than mTOR-dependent (Figure 2A).
For staining and microscopy, animals were dissected and fixed for 30 60 min in either Bouin's fixative (when GluRIIA or nc82 antibodies were used), or 4% paraformaldehyde (for all other immunolabeling).
For staining and microscopy, animals were manually dissected and fixed for 30 60 min in either Bouin's fixative (when Sec8 or GluRII antibodies were used), or 4% paraformaldehyde (for all other staining).
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To evaluate whether SM binding efficiency is limited by on- or off-rates, SNARE Sec17 Sly1 co-complexes were formed at 30°C for 60 min, washed in either 30°C buffer or 4°C buffer, and incubated an additional 60 min at the same temperature as the prior wash.
A robust induction of both Arc and c-fos mRNAs was observed in the hippocampus, with a peak at 60 min for Arc mRNA in either wild-type (Figure 5H) or hip-eEF-2K-tg mice (Figure 5I) and a peak at 30 min for c-fos mRNA in either wild-type (Figure 5J) or hip-eEF-2K-tg mice (Figure 5K).
Cells were initially rinsed twice with pre-warmed Opti-MEM and incubated for 60 min at 37°C in either Opti-MEM or one of four the incubation solutions.
BAL cells in 10 ml BAL fluid were pelleted by centrifugation (200 × g, 10 min), and resuspended in either 350 μl RLT buffer (Qiagen, Sweden) or 1 ml DMEM with 20% fetal calf serum, and stored at −70°C.
After addition of 50 μL radiotracer (>100,000 cpm), cells were incubated at 37°C for 120 min in triplicate with either 50 μL PBS with 0.5% BSA (unblocked) or 50 μL of 50 μM H-C*RRETAWAC*-OH in PBS/0.5% BSA (blocked), respectively.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com