Rabbit anti actin

The following antibodies were used in IHC and IF: guinea pig anti-insulin (DAKO, #A0564, 1:1,000), rabbit anti-glucagon (Phoenix, #H-028–05, 1:2,000), rabbit anti Ki67 (Abcam, #ab16667, 1:200), rabbit anti-cleaved caspase 3 (Cell Signaling, #9661, 1:200), rat anti-BrdU (Abcam, #ab6326, 1:200), mouse anti-Nkx6–1 (Hybridoma Bank, #F55A12, 1:100) rabbit anti-Mafa (Bethyl, #IHC-00352, 1:100), rabbit anti-Nkx2–2 (Sigma, #HPA003468, 1:100), rabbit anti-ALDH1A3 (Novus, #NBP2–15339, 1:100) and rabbit anti-gastrin (Millipore, #256A, 1:200). The following antibodies were used for western blot: rabbit anti-cleaved caspase 3 (Cell Signaling, #9661, 1:1,000), rabbit anti-adipsin (Santa Cruz, #sc-50419, 1:1,000), chicken anti-C3a (Abcam, #ab48581, 1:1,000), rabbit anti-Dusp26 (Invitrogen, #PA5–22013, 1:1,000), rabbit anti-actin (Cell Signaling, #8456, 1:1,000) and mouse anti-FLAG (Sigma, #A8592, 1:1,000). Recombinant C3a (R&D) was used at a concentration of 100 nM. NSC-87877 compound was dissolved in PBS and used at 20 μM concentration.

Cell lysates were collected using Protease arrest (GBiosciences, Maryland Heights, MO) in cell lysis buffer (Cell Signaling Technology, Danvers, MA). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermofisher, Waltham, MA). 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio–Rad, Hercules, CA) were loaded with 30 μg protein, electrophoresed and transferred to nitrocellulose (Pall Corporation, Ann Arbor, MI), and membranes were blocked for 60 minutes in Blocker casein in tris-buffered saline (TBS) (from Thermofisher, Waltham, MA). Blots were incubated with primary antibodies overnight in block buffer + Tween20 (1:1000) with rabbit anti-IκB and rabbit anti-actin (1:1000, antibodies all purchased from Cell Signaling, Danvers, MA). Blots were washed six times for 5 minutes each in TBS-Tween-20, followed by incubation with secondary antibody (1:10,000) for 60 minutes at room temperature (Cell Signaling, Danvers, MA). Size estimates for proteins were obtained using molecular weight standards from Bio–Rad (Hercules, CA). Blots were visualized and quantified using a LiCor Odyssey CLx Infrared imaging system (Lincoln, NE). After background subtraction, fluorescence intensity for the protein of interest was normalized to the signal intensity for the actin calculated relative to unactivated samples, using Image Studio 4.0 (LiCor).

Immunoblotting was performed as described (Sawamiphak et al., 2010 (link)). Mouse anti-PhosphoStat3 (MBL), rabbit anti-Stat3 (Santa Cruz), and rabbit anti-actin (Cell Signaling Technology) were used at a dilution of 1:500, 1:500, and 1:1000, respectively.