Polyacrylamide gels

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.

polyacrylamide gels of different stiffness levels were used for cell culture, as reported previously [39 (link)]. Briefly, coverslips were coated with a thin layer of gel containing a mixture of 3-10% acrylamide and 0.01%-0.3% bis-acrylamide, resulting in gels with stiffness levels of 2, 8 and 16 kPa. Polyacrylamide gel polymerization was promoted by the addition of 10% ammonium persulfate (1/100 volume) and tetramethylethylenediamine (3/1000 volume). The coverslips were washed twice for 20 minutes each in PBS on a rocker, and then were sterilized in a PBS solution for one hour with ultraviolet light. Subsequently, 50 μL of heterobifunctional sulfosuccinimidyl 6-(4′-azido-2′-nitrophe-nylamino) hexanoate was added and photoactivated for five minutes with ultraviolet light. After being rinsed with a PBS solution, the coverslips were coated with 10 μL/mL fibronectin for 1.5 hours and rinsed before cell seeding. The gels were soaked in serum-free culture medium for 24 hours before usage. All elements of the polyacrylamide gels were purchased from Solarbio (China).