Hatf00010

Cells or tissues were harvested, and 90 μg of total protein per sample was separated by gel electrophoresis and transferred to a hybridization nitrocellulose membrane (HATF00010, Merck, Darmstadt, Germany). The membrane was incubated overnight with primary antibodies (Table 1) at 4 °C. Before incubation with the fluorescence-conjugated anti-rabbit IgG secondary antibody (1:8000, 926-32211, LI-COR Biosciences, NE, USA), the membranes were washed with phosphate-buffered saline Tween (PBST) three times. Western blot bands were analyzed with an Odyssey imaging system (LI-COR, Inc., Lincoln, NE, USA).Table 1

Antibodies for western blot.

Anti-body	Company	Catalog Number	Dilution ratio
cGAS	Gene Tex	GTX02874	1:1000
STING	Gene Tex	GTX85266	1:1000
IRF3	Gene Tex	GTX54366	1:500
p-IRF3	Cell Signaling Technology	#29047	1:500
TBK1	Cell Signaling Technology	#38066 S	1:1000
p-TBK1	Cell Signaling Technology	#3504	1:1000
NLRP3	Cell Signaling Technology	#15101	1:1000
GSDMD	ABclonal Technology	A20728	1:500
GSDMD-N	ABclonal Technology	A17308	1:500
β-Actin	Gene Tex	GTX109639	1:3000

Pancreatic tissues were ground in liquid nitrogen and then sonicated (power 20%, 5 seconds on, 5 seconds off, total time 5 minutes) in lysis buffer (2% SDS, 10% glycerol, 10 mM Tris (pH 6.8) and 100 mM DTT). The tissue suspension was then centrifuged for 10 minutes (4°C, 12,000 rpm). The supernatant was removed and boiled for 10 minutes. Protein lysates were resolved on NuPAGE™ 4%‐12% Bis‐Tris Mini Protein Gel (#NP0336BOX; Invitrogen) and transferred to 0.45 μm nitrocellulose membranes (#HATF00010; Merck). Protein samples were normalized to GAPDH. Antibodies utilized are listed in Table 1. Imaging was taken using LI‐COR Odyssey CLx.

To determine the level of type I collagen secreted from control and CHX-treated MEFs, western blot analysis was performed on the cell lysate and conditioned media according to Shi
et al.
[17] (link). The samples were mixed with 4×SDS reducing sample buffer containing 40% glycerol, 250 mM Tris-HCl, pH 6.8, 8% sodium dodecyl sulfate, 0.04% bromophenol blue, and 20% 2-mercaptoethanol. The samples were then heated at 100°C for 10 min and subjected to 7.5% or 10% SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane (HATF00010; Merck Millipore, Billerica, USA), which was blocked with 5% BSA in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) at room temperature (RT) for 3 h and then incubated with anti-type I collagen antibody (0.4 μg/mL), anti-P4HA1 (1 μg/mL), anti-P4HA2 (1 μg/mL), or anti-actin antibody (0.2 μg/mL) overnight at 4°C. Bound primary antibodies were detected with HRP-conjugated rabbit anti-goat, goat anti-rabbit or anti-mouse IgG secondary antibodies (0.12 μg/mL). The amount of type I collagen was quantified by densitometry using ImageJ software (NIH) and was normalized to actin loading controls.