Lacrimal gland tissue was extracted with Radioimmunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime) mixed with phenylmethylsulfonyl fluoride (PMSF; Solarbio). Protein extracts were resolved via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; EPizyme, Shanghai, China) and then electrotransferred to a polyvinylidene difluoride membrane. After blocking, the membranes were probed with primary antibodies to AQP5 (1:2000, Abcam), α-SMA (1:2000, Abcam), GAPDH (1:3000, Kangchen, Shanghai, China), CHOP (1:500, Affinity Biosciences, Wuhan, China), Bax (1:1000, Affinity Biosciences), Bcl-2 (1:2000, Affinity Biosciences), Caspase12 (1:1000, Affinity Biosciences), and GRP78 (1:1000, Affinity Biosciences). Horseradish peroxidase–conjugated secondary antibodies were used, and the bands were developed using a Western Bright Electrochemiluminescence (ECL) substrate (Applygen, Beijing, China). GAPDH was used for normalization of expression [34 (link)].
Protocol detail
Western Blotting of Lacrimal Gland Proteins
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Antibodies
Bcl 2
Buffer
Chop
Gapdh
Grp78
Horseradish peroxidase
Lacrimal gland
Membranes
Phenylmethylsulfonyl fluoride
Polyvinylidene difluoride
Protein
Radioimmunoprecipitation assay
Sds page
Tissue
Corresponding Organization :
Other organizations : Qingdao University, People's Liberation Army 401 Hospital, Chinese People's Liberation Army, Shandong Eye Institute & Hospital
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Variable analysis
independent variables
- Lacrimal gland tissue extraction with RIPA Lysis Buffer and PMSF
- Protein expression levels of AQP5, α-SMA, GAPDH, CHOP, Bax, Bcl-2, Caspase12, and GRP78
- GAPDH was used for normalization of expression
- No positive or negative controls were explicitly mentioned.
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