Alexa fluor 488 dextran

In partial Rec8 degradation TRIM-Away experiments, TRIM21 expressing, metaphase II–arrested eggs were microinjected with 2 to 3 pl of Rec8 antiserum (1:30 to 1:50 dilution) (19 (link)) and Alexa Fluor 488 Dextran 10,000 molecular weight (MW) (to validate antibody microinjection by fluorescence imaging) (1:40 dilution; Molecular Probes, D22910) in 0.05% (v/v) NP-40–PBS. In complete Rec8 degradation TRIM-Away experiments, TRIM21-expressing, metaphase II–arrested eggs were microinjected with 2 to 3 pl of Rec8 antiserum (1:2 dilution) (19 (link)) and Alexa Fluor 488 Dextran 10,000 MW (1:40 dilution; Molecular Probes, D22910) in 0.05% (v/v) NP-40–PBS. In simultaneous Rec8 degradation and cytoskeletal manipulation experiments, all TRIM-Away microinjections were performed in M2 medium containing individual or a combination of cytoskeletal drugs as appropriate.

Dextran trafficking and fusion with CCVs was measured as described previously [47 (link)]. Briefly, HeLa cells were infected with mCherry-WT C. burnetii in six-well plates (5X10⁴ cells/well; two wells per condition). On the day before the indicated time points, cells were trypsinized, resuspended to 3X10⁵ cells/ml, and replated onto ibidi slides (9X10³ cells per channel). On a Nikon spinning disk confocal microscope (60X oil immersion objective) with Okolab Bold Line stage top incubator, CCVs were identified and marked using NIS elements (Nikon) prior to labeling with Alexa fluor 488 dextran (MW 10,000, Invitrogen) for 10 min in 10% RPMI. The cells were washed with PBS 5–6 times and replaced with 10% RPMI. Z-stacked confocal images were obtained for each CCV every 4 min for 28 min (t = 0 through 28; 8 time points). The mean dextran fluorescent intensity of an identical region of interest (ROI) within each CCV was quantified for each time point (ImageJ). The fold change of dextran fluorescent intensity over initial time point (t = 0) was plotted against time. At least 15 CCVs were imaged per condition for each of three independent experiments.