Alexa fluor 647 affinipure donkey anti goat

MEFs were purchased from ATCC (CRL-2752) and cultured according to their establish protocols. These MEFs were transduced with lentiviral particles from the described plasmids. Specifically, viruses were generated in HEK 293T/17 cells (CRL-11268; ATCC) by co-transfecting VSV-G, delta 8.9, and the indicated construct. 10 mM sodium butyrate was then added to the transfected cells 3 h post transfection for an overnight (∼16 h) incubation at 37°C, 5% CO₂. The transfection media containing sodium butyrate was removed the following day and the cells were washed with 1× PBS. Opti-MEM was then added back to the cells which were then incubated at 37°C, 5% CO₂. Viral supernatant was collected every 12 h (up to four collections), and the supernatant of all collections were pooled. MEFs were incubated overnight with fresh viral supernatants supplemented with 4 μg/ml polybrene and 10% FBS. Fresh MEF medium was then added to the cells after the virus was removed and selected with 10 μg/ml blasticidin or 1 µg/ml puromycin (or both). Antibodies used in this study include an anti-LMNB antibody (sc-6217, goat IgG; Santa Cruz Biotechnology, Inc.), Alexa Fluor 647 AffiniPure Donkey Anti-Goat (#705-606-147; Jackson Immunoresearch), and anti-biotin antibody (A150-109A; Bethyl Laboratories, Inc.).

Cells were prepared for immunofluorescence by plating on sterilized 25-mm round coverslips (German borosilicate glass #1.5; Harvard Apparatus) in six-well tissue culture dishes. Immunofluorescence was carried out as previously described (12 (link), 114 (link)). The nuclear lamina was visualized using an anti-LMNB antibody (sc-6217, goat IgG; Santa Cruz Biotechnology, Inc.) and Alexa Fluor 647 AffiniPure Donkey Anti-Goat (#705-606-147; Jackson Immunoresearch) for secondary detection. Biotinylation was detected using streptavidin-488 (#016-540-084, Alexa Fluor 488 Streptavidin; Jackson Immunoresearch). Immunofluorescence samples were mounted in SlowFade gold (Life Technologies). All imaging was performed on an inverted fluorescence microscope (AxioVision; Carl Zeiss) fitted with an ApoTome and camera (AxioCam MRm; Carl Zeiss). The objective lens used was a 63× apochromat oil immersion (Carl Zeiss) with an NA of 1.5 (Immersol 518; Carl Zeiss). All immunofluorescence was performed at room temperature on #1.5 coverslips. AxioVision software (Carl Zeiss) was used for image acquisition. Images were exported as TIFFs to (FIJI ImageJ, National Institutes of Health) for further analyses (115 (link)).