Mouse anti c myc mab 9e10

SDS-PAGE and immunoblotting onto PVDF membranes (Millipore) were performed as described (Lauber et al. 1997) . All antibodies were diluted in 5% (w/v) milk/13 Tris-buffered saline 1 0.1% (v/v) Tween 20. Antibodies and dilutions were as follows: mouse anti-c-Myc mAb 9E10 (Santa Cruz Biotechnology) 1:1000, mouse anti-GFP (Roche) 1:2500, rabbit anticalnexin (Agrisera) 1:2000, rabbit anti-AALP (anti-aleurain; Holwerda et al., 1990 [a gift from Inhwan Hwang]) 1:1000, rabbit anti-ARF1 (Agrisera) 1:2500, rabbit anti-SEC7 (Steinmann et al., 1999) 1:2500, mouse anti-LexA (Santa Cruz Biotechnology) 1:1000, POD-conjugated anti-HA (Roche) 1:4000, anti-mouse (Sigma-Aldrich) or anti-rabbit peroxidase-conjugated (Merck Millipore) or alkaline phosphatase-conjugated antibodies (Jackson Immuno Research) 1:10,000. Detection was performed with the BMchemiluminescence blotting substrate (Roche) and FusionFx7 imaging system (PeqLab). Image assembly was performed in Adobe Photoshop CS3, and Image Studio Lite Software (https://www.licor.com/bio/imagestudio-lite/) was used for quantification of relative protein amounts.

Four-to 6-d-old seedlings were incubated on 24-well cell culture plates for 1 h in 50 mM BFA (Invitrogen, Thermo Fisher Scientific) containing liquid growth medium (half-strength MS medium and 1% [w/v] Suc, pH 5.8) at 23°C and then fixed for 1 h in 4% (w/v) formaldehyde in microtubulestabilizing buffer at room temperature. Whole-mount immunofluorescence staining was performed manually as described (Lauber et al., 1997) or with an InsituPro machine (Intavis; Müller et al., 1998) . All antibodies were diluted in 13 PBS. The following antisera were used for immunofluorescence staining: mouse anti-c-Myc mAb 9E10 (Santa Cruz Biotechnology) diluted 1:600; rabbit anti-ARF1 (Agrisera) diluted 1:1000; rabbit anti-AtgCOP (Agrisera) diluted 1:1000; anti-mouse Alexa488 (Invitrogen) and antirabbit CY3 (Dianova)-conjugated secondary antibodies diluted 1:600. Nuclei were stained with 49,6-diamidino-2-phenylindole (1:600 dilution).