Loading buffer

Proteins from cell cultures were extracted with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitors unless otherwise noted. Loading buffer (Life Technologies) was added, and samples were heated to 95 °C for 5 min, subjected to electrophoresis on 4–12% NuPAGE Bis-Tris gels (Life Technologies), transferred to nitrocellulose membranes (Life Technologies), subjected to western blot analysis and visualized using the Odyssey CLx system (LI-COR Biosciences). The primary antibodies were anti-DSP (MAB9080, R&D Systems) (1:2000 dilution), anti-PPL (ab131269, Abcam) (1:2000 dilution) and anti-FLAG (F3165, Sigma-Aldrich) (1:2000 dilution). Anti-GAPDH antibodies (ab181602, Abcam; TA802519, Origene) (1:2000 dilution) and anti-HSP90 antibodies (ab13495, Abcam; TA500494, Origene) (1:2000 dilution) were used as loading controls. The secondary antibodies used were IRDye 680RD goat anti-mouse (LI-COR Biosciences) (1:10,000 dilution) and IRDye 800CW goat anti-rabbit (LI-COR Biosciences) (1:10,000 dilution). Blots were quantified using the Image Studio software (LI-COR Biosciences).

Whole cell lysates from S2VP10L, MiaPaCa-2, S2013, and SCC-1 cells were collected to determine IGF1R expression. The cells were plated at a density of 5x10⁵ cells per well in a 6-well plate 24 h prior to protein harvest. Cells were then lysed in a solution containing 1 % NP-40, 1 % protease inhibitor, and 1 % phosphatase inhibitor (Thermo) in deionized water. The lysates were centrifuged at 13,000×g for 10 min. Total protein concentration was determined via Bradford assay (Bio-Rad, Hercules, CA, USA).
Approximately 50 µg of total protein was dissolved in deionized water, loading buffer, and reducing agent (Life Technologies, Carlsbad, CA, USA). Proteins were separated using NuPage 4–12 % Bis–Tris gel and transferred onto a nitrocellulose membrane by iBlot (Life Technologies). The membrane was blocked in blocking buffer (Li-Cor) for 30 min and then incubated overnight at 4 °C with IGF1R antibody (Abcam, Cambridge, England) at a concentration of 1 µg/mL and β-Actin antibody (Thermo) at a concentration of 1:3000. The membrane was then washed 3× with TBS (20 mM Tris–HCl, 150 mM NaCl in diH₂O) for 10 min each, incubated with donkey anti-mouse IRDye 680RD and donkey anti-rabbit IRDye 800CW secondary antibodies (Li-Cor) for 1 h, washed 3× with TBS for 10 min each, and scanned using Li-Cor Odyssey infrared scanner. Dosimetry was performed using Li-Cor software.

RIPA lysate (Pierce) was added to the tissues/cells to obtain the protein that was mixed with the loading buffer (Life Technologies) at a ratio of 3:1, and boiled at 100°C for 8‐10 min. Protein samples were added to the gel well and the target protein was separated by electrophoresis. The protein was transferred to a PVDF membrane (Millipore), and non‐specific antigens blocked with 5% skim milk. The membranes were incubated with corresponding primary antibodies: anti‐CD9 (1:500; Proteintech), anti‐CD63 (1:500; Proteintech), anti‐CD81 (1:500; Proteintech), anti‐NEDD8 (1:500; CST), anti‐cullin 1 (1:500; Abcam), anti‐IκB (1:500; CST), anti‐NF‐κB (1:500; Abclonal), anti‐P‐NF‐κB (1:500; Abclonal), anti‐PCNA (1:500; Abcam), and anti‐β‐actin (1:500; SAB) overnight at 4°C. After washing with 1× TBS/T buffer, the membranes were incubated with the secondary antibody for 30 min at 37°C. Pictures were taken with a chemical gel imaging system (GE).