Sc 562

Tissues were homogenized in hypotonic buffer with Triton X-100 while using metal beads in a Tissue Lyser (Qiagen, Hilden, Germany) as previously described [19 (link)]. 40–50 μg of total protein was resolved per lane by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were probed with specific antibodies against GRK2 (sc-562, Santa Cruz Biotechnology, Dallas, TX, USA), β-Arrestin 1 and 2 [20 (link)], anti-pAkt (Ser473 #9271, Cell Signalling, Danvers, MA, USA), total Akt (#9272 Cell Signalling), β-Actin (127M4866V, Sigma, San Luis, MO, USA), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, sc-32233, Santa Cruz), anti-phospho-tyrosine (#61-5800, Invitrogen, Carlsbad, CA, USA), anti-insulin receptor β subunit (sc-57342, Santa Cruz), anti IGF1R (#9750, Cell Signalling), and α-Tubulin (sc-53030, Santa Cruz). Immunoreactive bands were visualized while using enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) or the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). Films were scanned with a GS-700 Imaging Densitometer and then analyzed with Quantity One Software (Bio-Rad, Hercules, CA, USA), or using an Odyssey Classic reader and the Odyssey software package 3.0 (Li-Cor Biosciences).

For whole-pancreas lysates, complete pancreatic tissue was homogenized in 1.5 ml of hypotonic lysis buffer as previously described for cardiac tissue [93 (link)] using metal beads in a Tissue Lyser (Qiagen) with two 2-min pulses of 1/30 s speed. Pancreatic islets and cultured cells were ruptured in lysis buffer by bath sonication and centrifuged before protein concentration was measured in the supernatant by ABC (Bio-Rad) or Lowry standard methods.
40–50 μg (whole pancreas) or 10–30 μg (cell or islets) of total protein lysates per lane were resolved by SDS-PAGE and transferred to nitrocellulose or PVDF membranes. Blots were probed with specific antibodies against GRK2 (sc-562, Santa Cruz Biotechnology, Batch number: J0615, 1:1000), β-Actin (Sigma Sigma A5441, RRID: AB_476744, Batch number: 0000088070, 1:2000), GAPDH (14C10, Cell Signaling, RRID: AB_10693448, 1:1000) and developed using enhanced chemiluminescence (ECL; Amersham Biosciences) or the Odyssey Infrared Imaging System (Li-Cor Biosciences). Films were scanned with a GS-700 Imaging Densitometer and analyzed with Quantity One Software (Bio-Rad), or using an Odyssey Classic reader and the Odyssey software package 3.0 (Li-Cor Biosciences).

Briefly, the spinal dorsal horn of the L4-L6 segments was lysed in RIPA Lysis Buffer (100 μl/g, Beyotime Biotechology, P0013B), supplemented with protease inhibitors (Beyotime Biotechology, ST506). The lysate was centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was obtained. Samples were separated with 10% acrylamide gels and then transferred onto polyvinylidene fluoride membranes by using SDS-PAGE. After blocking with 5% skim milk in tris-buffered-saline with tween (TBST) (20 mm Tris–HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween-20), the membranes were incubated with rabbit anti-GRK2 (1:300, Sc-562, Santa Cruz), rabbit anti-GAPDH (1:10,000, HRP-60004, ProteinTech) or anti-β-actin (1:10,000, HRP-60008, ProteinTech) overnight at 4 °C in a shaking incubator, then washed in TBST before incubated with appropriate HRP-conjugated secondary antibody (1:10,000) for 2 h at room temperature. The objective band were detected using an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK) and analyzed using ImageJ software (version 1.47).