Protein extracts were transferred to polyvinylidene difluoride membranes as previously described (3 (link)). Membranes were blocked with Odyssey Blocking Buffer in tris-buffered saline (LI-COR) and incubated overnight at 4°C with the following primary antibodies: Hspa5 (1:2000; Proteintech, 11587-1-AP), Pdia6 (1:2000; Abcam, ab11432), Biotin Ligase Epitope Tag (for Tavi-tag detection, 1:1000; Abcam, ab106159), PSD95 (1:5000; Thermo Fisher Scientific, 6G6-1C9), hemagglutinin (HA) (1:2000; Millipore Sigma), phospho-tau AT8 (1:5000; BioLegend, 806503), actin (1:10,000; Thermo Fisher Scientific), ATF4 (1:500; Thermo Fisher Scientific, PA5-27576), ATF6 (1:500; Thermo Fisher Scientific, PA5-114886), and phospho-IRE1α (1:500; Thermo Fisher Scientific, PA5-85738). Membranes were washed and incubated with appropriate IRDye immunoglobulin G (IgG) secondary antibodies, including anti-rabbit IRDye 800LT (1:5000; LI-COR) and anti-mouse IRDye 680CW (LI-COR). Images were acquired using the Odyssey Infrared Imaging System (LI-COR). Quantification of Western blot bands was performed using Image Studio Lite v.5.2 (LI-COR).
Protocol detail
Western Blot Analysis of Protein Expression
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Odyssey Blocking Buffer
Hspa5
Pdia6
PSD95
actin
phospho-IRE1α
anti-rabbit IRDye 800LT
anti-mouse IRDye 680CW
Odyssey Infrared Imaging System
Image Studio Lite v.5.2
Actin
Antibodies
Atf4
Atf6
Biotin ligase
Buffer
Epitope
Hemagglutinin
Hspa5
Immunoglobulin g
Ire1α
Membranes
Mouse
Polyvinylidene difluoride
Protein
Rabbit
Saline
Western blot
Corresponding Organization : University of Iowa
Other organizations : King's College London
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Variable analysis
independent variables
- Primary antibodies used for Western blot analysis: Hspa5, Pdia6, Biotin Ligase Epitope Tag, PSD95, hemagglutinin (HA), phospho-tau AT8, actin, ATF4, ATF6, and phospho-IRE1α
- Western blot band quantification
- Protein extracts were transferred to polyvinylidene difluoride membranes
- Membranes were blocked with Odyssey Blocking Buffer in tris-buffered saline
- Membranes were incubated overnight at 4°C with primary antibodies
- Membranes were washed and incubated with appropriate IRDye immunoglobulin G (IgG) secondary antibodies
- Images were acquired using the Odyssey Infrared Imaging System
- Quantification of Western blot bands was performed using Image Studio Lite v.5.2
- No positive or negative controls were explicitly mentioned
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