Genomic DNA from N. punctiforme was used as a template for the hupSL promoter-hupS- and the hupSL intergenic region-hupL-containing DNA fragments. The gfp-modified hup-operon PCR product was cloned into the pBluescript II SK+ plasmid (Stratagene) before subcloning into pSUN119 (Argueta et al., 2004) using SmaI and SacI (Fermentas), generating plasmid pSHG. The complete sequence of the gfp-modified hup-operon is available (Supporting Information). Finally, pSHG was transferred into N. punctiforme by electroporation

and positive clones were selected as described previously (Holmqvist et al., 2009), using 10 μg mL−1 neomycin (creating INCB024360 the SHG culture). The GFP and phycobilisome/photosystem II emission of WT, SHG, and GFP control [N. punctiforme containing the pPMQAK1-Ptrc1O-GFP plasmid (Huang et al., 2010)] cultures were examined as described previously (Cardona et al., 2009). Nonconfocal differential interference contrast (DIC) reference images were produced on a separate channel. GFP was excited using 488-nm laser light and emission was detected from 500 to 540 nm. Confocal microscopy settings, laser effects and PMT voltages were kept identical to enable comparison of GFP fluorescence signal strength for studying the Small Molecule Compound Library cellular localization of GFP, but not for studying

the subcellular localization. Overlay images were produced from confocal red autofluorescence, confocal GFP fluorescence, and nonconfocal DIC images using the las af software (Leica). Image processing was performed using Photoshop Hydroxychloroquine chemical structure CS4 Extended (Adobe Systems). The red autofluorescence (in magenta) was enhanced for clarity.

The GFP fluorescence was not edited. Heterocyst isolations were performed as in our previous work on N. punctiforme (Cardona et al., 2009; Ow et al., 2009), using protocols originally established by (Almon & Böhme, 1980). Chlorophyll a measurements were carried out as reported previously (Holmqvist et al., 2009). Proteins from isolated heterocysts were extracted as described (Ow et al., 2009; Agervald et al., 2010) using denaturing buffer [50 mM Tris-HCl, pH 7.8, 14.2 mM β-mercaptoethanol, 2% sodium dodecyl sulphate (SDS)] or native buffer, (25 mM BisTris, pH 7, and 20% glycerol) supplemented with Complete Mini, EDTA-free protease inhibitor cocktail tablets (Roche). The protein concentrations were determined using colorimetric Bradford protein assay (Bio-Rad Laboratories) and 50 μg total proteins were separated on 12% SDS-PAGE gels run at 200 V. To examine whether HupS–GFP forms a complex with HupL, attempts were made to extract HupS–GFP under native conditions, with no success. To examine the solubility of HupS–GFP, proteins from equal amounts of SHG cultures were extracted as above, but using buffers containing no detergents, mild nonionic detergents (0–2% Triton X-100 or 0–5% dodecyl maltoside), or strongly denaturing additives (7 M urea and 2 M thiourea) (see Supporting Information, Fig. S1, for details).