Kpl lumiglo reserve chemiluminescent substrate kit

3x10⁶ BMDM were seeded in 6-well plates and pretreated with berberine (30 µM) or vehicle (DMSO 0.1%) overnight. BMDM were infected H37Rv for 30, 60 and 120 minutes and washed with cold PBS before lysing with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) including cOmplete Protease inhibitor and PhosSTOP phosphatase inhibitor cocktail (Roche). Cell lysate protein content was determined using the BCA Protein Assay Kit (ThermoFisher). 30 µg of protein was loaded to 10% resolving acrylamide gel and wet WB was performed onto a nitrocellulose membrane. The membrane was probed with either SAPK/JNK Antibody, Phospho-SAPK/JNK (Thr183/Tyr185) (G9), NFκB p65 (D14E12), Phospho-NFκB p65 (Ser536) (93H1) (Cell Signaling Technology) or GAPDH (Santa Cruz Biotechnology) primary antibodies and either with goat anti-rabbit IgG H&L (HRP) pre-absorbed or goat anti-mouse IgG H&L (HRP) pre-absorbed (Abcam) secondary antibodies. Immunoblots were developed using the KPL LumiGLO ^® Reserve Chemiluminescent Substrate Kit (SeraCare) on the iBright FL1000 Imaging System (Thermo Fisher).

Primary salivary gland epithelial cells were generated from ΔNp63^fl/fl (control) and UBC^CreERT2;ΔNp63^fl/fl (ΔNp63KO) mouse submandibular glands and seeded on plastic 6 well plates (10⁶ cells/well) in CnT-Prime medium (CELLnTEC) and cultured at 37 °C in 5% CO₂. The cells were treated with activated tamoxifen ((Z)-4-Hydroxytamoxifen, 4-OHT, Sigma-Aldrich) 11 days after plating in order to knock down ΔNp63. Three days after tamoxifen treatment, the cells were washed with PBS, lysed in RIPA buffer containing a protease inhibitor cocktail (G-Biosciences), and subjected to western blot analysis. Protein concentration was determined by using the Bio-Rad Bradford protein assay. The protein samples were separated by SDS-PAGE and transferred to a PVDF membrane and blocked with 5% non-fat dry milk in Tris Buffered Saline with Tween-20 (TBST). For primary antibodies, p63 (1:10,000, Cell Signaling Technology), SMA (1:10,000, Sigma-Aldrich), and β-actin (1:10,000, Cell Signaling Technology) were used. KPL LumiGLO Reserve Chemiluminescent Substrate kit (Sera care) was applied to the membrane and the ChemiDoc MP Imaging System (Bio-Rad) was use for detection. Densitometry (measurement of band intensity) was performed by using Image J (NIH) and p63 and SMA expression was normalized by β-actin. (n = 3).

Western blot analysis was performed as previously described (29 (link)). Cell lysate protein content was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts) after which a total of 20-40 µg of protein was used to determine expression patterns. The membrane was probed with either p38 MAPK (D13E1) XP^®, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP^®, SAPK/JNK Antibody, Phospho-SAPK/JNK (Thr183/Tyr185) (G9), NFκB p65 (D14E12) XP^®, Phospho-NFκB p65 (Ser536) (93H1) XP^® (all from Cell Signaling Technology, Danvers, Massachusetts) NFκB p50/p105 (Clone 1N19, ZooMAb^®) (Sigma Aldrich, St. Louis, Missouri) or GAPDH (Sant Cruz Biotechnology, Dallas, Texas) primary antibodies and captured using either goat anti-rabbit IgG H&L (HRP) pre-absorbed or goat anti-mouse IgG H&L (HRP) pre-absorbed (both from Abcam, Cambridge, UK). Immunoblots were developed using the KPL LumiGLO^® Reserve Chemiluminescent Substrate Kit (SeraCare Life Sciences, Milford, Massachusetts) on the iBright FL1000 Imaging System (Thermo Fisher Scientific, Waltham, Massachusetts). Densitometry analysis was performed using the built-in iBright FL1000 Imaging System after which each band was normalized to GAPDH.