Total proteins from A549 cells were isolated using RIPA lysis buffer (Aspen Biotechnology, Wuhan, China). BCA kit was performed to quantify the concentration of proteins. After that, proteins (10 μg/lane) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Next, PVDF membranes were incubated with primary antibodies anti-KLF1 (Abcam, Cambridge, MA, USA; 1:1000; Rabbit), anti-E-cadherin (1:1000; Rabbit), anti-N-cadherin (1:1000; Rabbit), anti-β-catenin (1:1000; Rabbit), anti-cleaved caspase 3 (1:1000; Rabbit), anti-Bcl-2 (1:1000; Rabbit), or anti-β-actin (1:1000; Rabbit) at 4°C overnight. Subsequently, PVDF membranes were incubated with the secondary antibodies (1:5000; Goat Anti-Rabbit) for 1 h. Then, the protein bands were observed using an efficient chemiluminescence (ECL) kit according to the previous literature [35 (link)]. β-actin was viewed as an internal control.
Protocol detail
Protein Expression Analysis in A549 Cells
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A549 cells
Actin
Anti antibodies
Antibodies
Bcl 2
Buffer
Cadherin
Cadherin 1
Caspase 3
Chemiluminescence
Goat
Klf1
Membranes
Protein
Pvdf
Rabbit
Ripa
Sds page
β catenin
Corresponding Organization : Hangzhou Medical College
Other organizations : Zhejiang University
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Variable analysis
independent variables
- RIPA lysis buffer (Aspen Biotechnology, Wuhan, China)
- Protein concentration
- Protein expression levels of KLF1, E-cadherin, N-cadherin, β-catenin, cleaved caspase 3, Bcl-2
- Total protein amount (10 μg/lane)
- Primary antibodies (anti-KLF1, anti-E-cadherin, anti-N-cadherin, anti-β-catenin, anti-cleaved caspase 3, anti-Bcl-2, anti-β-actin)
- Secondary antibody (Goat Anti-Rabbit, 1:5000)
- Efficient chemiluminescence (ECL) kit
- β-actin as internal control
- Not explicitly mentioned
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