Goat anti mouse igg hrp

For in-cell CRABP1–CaMKII assays, cell lysates were separated on 9% (v/v) SDS polyacrylamide gels and transferred onto 0.45-µm PVDF membranes (Millipore Sigma Cat. IPVH00010). For His pull-down assays, reactions were separated on 10% (v/v) SDS polyacrylamide gels and transferred on a 0.45-µm PVDF membrane. Primary antibodies and their dilutions used include anti-p-CaMKII (cat #: 127,165, 1/1,000) from Cell Signaling-Danvers, MA, United States, anti-GFP (cat #: SC-9996, 1/1,000) from Santa Cruz Biotechnology, anti-β-Actin (cat #: SC-47778, 1/1,000) from Santa Cruz Biotechnology-Dallas, TX, United States, anti-FLAG from Sigma (cat#: F3165, 1:1,000), and anti-His (Cat # sc-8036, 1:1,000) from Santa Cruz Biotechnology-Dallas, TX, United States, and secondary antibodies used include goat anti-mouse-IgG-HRP (cat #: GTX26789, 1/5,000) from GeneTex, Irvine, CA, United States and goat anti-rabbit-IgG (cat #: 11–035-144, 1/2000) from Jackson ImmunoResearch, Ely, United Kingdom.
WesternBright ECL substrate was used for chemiluminescent detection of Western blot signals (Advansta Cat # K-12045-D50) A Bio-Rad ChemiDoc Imager, Hercules, CA, United States (cat #: 17001402) was used to collect images.

Cells were sonicated or homogenized in RIPA buffer (20 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP‐40, 0.1% SDS, and 1% sodium deoxycholate). After being clarified by centrifugation at 12 000 g for 15 min at 4 °C, the supernatant was collected for Bradford protein assay. Western blotting was performed as previously reported.^[58 (link)
^] Briefly, equal molarity of protein extracts was loaded and separated in an SDS–PAGE, and transferred to a PVDF membrane. After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ˚C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

ARPE-19 cells were seeded at a density of 4 × 10⁵ in 6 cm dishes. After 24 h, cells were treated with 0.5 µM G570 and 150 lux blue light. After 24 h, cells were harvested in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, and 1% w/v Nonidet P-40) and placed on ice. Collected cells were centrifuged at 14,000 g for 10 min. Total protein was collected, and the concentration was measured using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA). Total proteins were isolated by SDS-PAGE electrophoresis (Bio-Rad Laboratories, CA, USA) and transferred onto PVDF membranes (Millipore Sigma, Burlington, MA, USA). After blocking with 5% w/v BSA (Sigma-Aldrich, Dublin, Ireland) for 40 min, the membranes were incubated with primary antibodies against 1:500 VEGFA-189 (Novus Biologicals, Centennial, CO, USA), 1:500 VEGFR2 (Novus Biologicals, CO, USA), 1:1000 ERK (Cell Signaling, Danvers, MA, USA), 1:1000 FAK (Abcam, Cambridge, UK), 1:1000 p38 (Cell Signaling, MA, USA), 1:2000 Akt (Cell Signaling, MA, USA), and 1:5000 eNOS (BD, Franklin Lakes, NJ, USA). Then, the membranes were washed twice with TBST and probed with 1:5000 goat anti-rabbit IgG (HRP) or 1:5000 goat anti-mouse IgG (HRP) (GeneTex, Irvine, CA, USA). The expression of these proteins was detected with HRP substrate luminol reagent by a BioSpectrum^® 500 Imaging System (UVP, Upland, CA, USA).