Cells were washed once with ice-cold PBS and lysed in 100 µl RIPA lysis buffer (Solarbio, #R0010) with PMSF and Cocktail. Lysate was extracted with SDS-PAGE loading buffer (Solarbio, #P1040) for 5 min at 100°C. Proteins were transferred to Immobilon-FL 0.45 µm PVDF membranes (Millipore) and subjected to immunoblot analysis with following antibodies:PD-L1 (1:1000, CST, #13684 S), phospho-ROS1 (1:1000, CST, #3078 S), ROS1 (1:1000, CST, #3266 S), p-ERK (1:1000, CST, #9106 S), ERK (1:1000, CST, #4695 S), p-STAT3(1:1000, CST, #9145 S), STAT3 (1:1000, CST, #9139 S), p-AKT (1:1000, CST, #4060 S) and AKT (1:1000, CST, #4685), β-actin (1:5000, ZSGB-BIO, #TA-09). Bio-Rad ChemiDocTM MP imaging system or LI-COR Odyssey Platform was used for western blot detection with either HRP-conjugated or IR dye secondary antibodies, respectively. Flow cytometry antibodies for PD-L1 were purchased from BD Biosciences, conjugated with PE-CY7 (BD, #558017) or APC (BD, #563741).
Immobilon fl 0.45 m pvdf membranes
Manufactured by Merck Group
Immobilon-FL 0.45 µm PVDF membranes are a type of laboratory equipment used for protein and nucleic acid transfer and detection. They are made of polyvinylidene fluoride (PVDF) and have a pore size of 0.45 micrometers. These membranes are designed for use in various blotting techniques, such as Western blotting and Northern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Sds page loading buffer, by Solarbio (1 mentions)
Odyssey platform, by LI COR (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
2 protocols using immobilon fl 0.45 m pvdf membranes
1
Western Blot Analysis of Cell Signaling
2
Quantitative Western Blot of α-Synuclein
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1×106 NPCs (passages 19, 23 and 27 were lysed on ice in 250 µl 1x RIPA buffer (Sigma #R0278). Protein concentration was determined with a BCA Assay (Pierce #23235). 30 µg protein was resolved by SDS-PAGE on NuPAGE 4–12% Bis-Tris pre-cast gels (Life Tech.), transferred onto Immobilon-FL 0.45 µm PVDF membranes (Millipore, #IPFL10100) and detected with the Li-COR Odyssey Western blotting Kit I or II (LI-COR Biosciences P/N 926-31081/2). Immuno-reactive bands were detected and quantified with the LI-COR ODYSSEY infrared imager. The polyclonal anti-α-syn specific antibody (1/300) was from Millipore (#AB5334P). HSP90 (#4874), β-tubulin (#2146) at and cytochrome c (#4272) all used at 1/1000 were from Cell Signaling Technologies.
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