Anti bip2

N. benthamiana leaves expressing NET3B-GFP were used for total microsomal fraction isolation. Approximately 0.1 g of leaf tissue was homogenized in 12% (w/v) sucrose buffer containing 50 mM Tris hydrochloride at pH 7.6), 100 mM sodium chloride and 5 mM EDTA. Ultracentrifugation was performed at 55000 rpm using a Beckman TLA-100 rotor for 60 min. Both total microsome pellet and supernatant were mixed with SDS buffer, fractionated by SDS-PAGE and subsequently subjected to western blotting. For immunoblotting, primary antibodies of anti-GFP (Abcam), anti-BIP2 (Agrisera) and anti-actin (C4, Millipore) were used at 1:1000, 1:1000 and 1:500, respectively. Horseradish peroxidase-conjugated secondary antibody and ECL reagent (GE Heathcare) were used for developing the membrane.

A GH3:GUS reporter line [50 (link)] was used as the WT moss strain. Spot cultures were grown as described previously [61 (link)], and tissue for genetic analysis was prepared as in [50 (link)]. All lines were stored in the International Moss Stock Center (http://www.moss-stock-center.org; see Supplemental Information).
For immunolocalizations, tissue was grown for 4 weeks in continuous light, fixed in 3:1 methanol acetic acid, dehydrated, and embedded in PEG 1600. Eight-micrometer sections were interrogated with anti-maize PIN antibodies [55 (link)] at a 1/150 dilution and anti-BIP2 (Agrisera) at a 1/50 dilution. DyLight 594 and DyLight 405 were used as secondary antibodies at a 1/300 dilution.
pin disruptants were generated and screened for insertion as described in Supplemental Information.
GUS staining was carried out as elsewhere [32 (link)]. Light micrographs were compiled using a Keyence VHX-1000 series microscope with 50× and 200× objectives. Confocal imaging was undertaken as previously described [61 (link)], except for immunolocalizations; a Leica TCS 5 was used, with excitation from the Diode 405 and HeNe 594 laser lines, and emission was collected at 410–480 nm and 600–670 nm.

For high-pressure freezing, root tips were excised from the wild-type Arabidopsis and overexpression lines, and briefly washed in Murashige and Skoog medium. Following a subsequent washing step in hexadecene, root tips were immediately frozen in a high-pressure freezer (HPF010; Bal-Tec). Freeze substitutions were performed in an AFS freeze substitution unit (Leica) in dry acetone supplemented with 0.1% uranyl acetate at −85 ℃ for 3 d before slowly being warmed to −35 ℃ for a period of 18 h. Samples were embedded in Lowycryl HM20, using gelatin capsules. The resin was polymerized with constant UV light for 2 d at −35 ℃ and an additional 3 d at 22 ℃. Thin sections were incubated with anti-BiP2 (Agrisera) and anti-GFP antiserum at a primary dilution of 1:200, followed by incubation with 10 nm gold-coupled secondary antibodies (Biocell GAR10 1:50), at a dilution of 1:30. Aqueous uranyl acetate/lead citrate post-stained sections were examined in a Philips CM10 transmission electron microscope operating at 80 kV.