Pvdf membrane

The oil body was diluted with PBS to 5 μg/µL, added 1 × loading buffer and the suspension was heated at 90 °C for 10 min. The oil body suspension was analyzed by 15% polyacrylamide gels (Beijing solarbio science & technology Co., Ltd., China). One of the gels was dyed by Coomassie Brilliant Blue (CBB) (BeiJing DingGuo ChangSheng Biotechnology Co., Ltd., China) staining method and decolored for 24 h with decolorization solution. Another gel was used to transfer protein to a PVDF membrane (BeiJing DingGuo ChangSheng Biotechnology Co., Ltd., China). It was blocked overnight with 20 mL of TBST buffer (pH = 8.0) (Beijing solarbio science & technology Co., Ltd., China) including 0.05% Tween-20 (MYM Biological Technology Co., Ltd., China) and 5% nonfat dry milk (BD Difco, USA). The blocked PVDF membrane was washed 4 times (8 min each time) with TBST buffer and incubated with a rabbit anti-hEGF polyclonal antibody (1:2000, Abcam, ab9605, Lot: GR11004-41 USA) followed by the secondary antibody which is goat anti-rabbit IgG/AP antibody (alkaline phosphatase-conjugated, Promega, S3738, Lot: 00001473 46, USA) [17 (link)]. The oleosin-hEGF-hEGF protein accumulation data in safflower seeds was analyzed with Quantity One software.

For western‐blotting, HeLa cells were plated in 12‐well plates and transfected with 1 µg plasmid. After 48 h transfection, cells were washed with PBS and lysed in 50 µL 1 × SDS loading buffer (50 mm Tris‐HCl pH 6.8, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 1% beta‐mercaptoethanol) at room temperature for 10 min. The cell lysate was collected and then boiled at 95 °C for 10 min. The appropriate amount of protein was loaded onto SDS PAGE gels. The separated proteins were transferred onto a PVDF membrane (Millipore) in an ice‐bath for 2 h. Then the PVDF membrane was blocked in 5% (w/v) BSA (Beijing Dingguo changsheng Biotechnology Co.,Ltd, FA016) in TBST (Tris‐buffered saline, 0.1% Tween 20) at room temperature for 1 h. The blot of protein was stained as indicate for at least 12 h at 4 °C. The blot was washed four times with TBST at room temperature for 5 min each, then stained with 1:5000 HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG(H+L) (Proteintech, SA00001‐2) or HRP‐conjugated Affinipure Goat Anti‐Mouse IgG(H+L) (Proteintech, SA00001‐1) in 5% BSA (w/v) in TBST for 1 h at room temperature. The blots were washed four times with TBST at room temperature for 5 min each time and imaged on Molecular Imager ChemiDocTM XRS+ Imaging System (Bio‐Rad) after incubation with Rhea ECL (US Everbright, Inc).