Horseradish peroxidase conjugated secondary antibody

The samples were homogenized and total proteins extracted using a radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China). A BCA kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. The proteins were loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels (10%) (Aspen), transferred onto nitrocel-lulose membranes (Pall Corp., New York, NY, USA), and blocked in non-fat dry milk (5%) at room temperature to set for 2 h. The membranes were incubated overnight at 4°C with primary antibodies against Ang-1 (1:1,000; ab102015; Abcam), p-Tie-2 (1:1,500; sc-9026; Santa Cruz Biotechnology, Inc.), α-SMA (1:2,000; ab32575), collagen type IV (1:1,500; ab6586) and GAPDH (1:5,000; ab37168) (all from Abcam). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Aspen) for 1 h, and finally the membranes were detected using an enhanced chemiluminescence substrate (Beyotime Institute of Biotechnology).

Total protein was extracted from the cells and quantified using a BCA Protein Assay Kit (AS1086, ASPEN Biotechnology). The proteins were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA) at 200 mA (constant current). The membranes were blocked with skimmed milk and incubated with primary antibodies against Grp78, ATF4, CHOP, PERK, eIF-2α, BAX, Bcl-2, p4E-BP1, and pT389-S6K overnight at 4°C using an incubator shaker. Details of the primary antibodies used are shown in Table 2. The loading control was β-actin. Tris-buffered saline containing 0.1% Tween-20 (TBST) was used for washing (3 washes of 5 min each). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000; ASPEN) for 30 min. An ECL kit (Solarbio Biotechnology Co., Ltd., Beijing, China) was used for visualization of the bands at 25°C and the bands were imaged using a Canon Chemiluminescent Imaging System (LiDE110, Canon, Japan).Table 2

Primary antibodies used for western blotting.

Table 2

Name	Dilution ratio	Article number	Manufacturers
Grp78	1:2000	11587-1-AP	Proteintech
ATF4	1:500	DF6008	Affbiotech
CHOP	1:2000	15204-1-AP	Proteintech
PERK	1:500	AF5304	Affbiotech
eIF-2α	1:1,000	AF6087	Affbiotech
BAX	1:5,000	50599-2-Ig	Proteintech
Bcl-2	1:3,000	26593-1-AP	Proteintech
p4EBP1	1:2,000	AF3432	Proteintech
pT389-S6K	1:1,000	9234	CST
β-actin	1:10,000	Ab181602	Abcam