Irdye 680lt goat anti mouse igg h l

THP‐1 cells pellets were extracted in RIPA lysis buffer (Beyotime, China) containing protease inhibitor cocktail (Calbiochem, USA). Protein concentrations were determined by a BCA kit (Pierce, USA). Equal amounts of cell extracts were electrophoresed on sodium dodecyl sulfate‐polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked in 5% non‐fat dried milk in TBS‐T at room temperature for 2 hours and incubated with indicated primary antibodies at 4°C overnight. The primary antibodies used in the study were: anti phospho‐NF‐кB (Ser468) (1:1000, #3039, Cell signaling technology, USA), anti‐TNFα (1:1000, MAB610, R&D systems, USA), and anti β‐actin (1:1000, #3700, Cell signaling technology, USA). The membranes were washed three times with TBS‐T for 10 min each time, and then incubated with IRDye‐680LT Goat Anti‐Mouse IgG (H+L) (Li‐Cor, USA) or IRDye‐800CW Goat Anti‐Rabbit IgG (H+L) (Li‐Cor, USA) (1: 10,000 in TBS‐T) at room temperature for 1 hour. After washing in TBS‐T for another three times, the membranes were analyzed by the Odyssey infrared imaging system (Li‐Cor, USA).
Levels of IL‐1β were determined in EC supernatants by an ELISA assay kit (DLB50, R&D Systems, USA) according to the manufacturer's instruction.

The following primary antibodies were used for detection by immunoblot: rabbit anti‐mGFP (Rocha et al, 2009), anti‐mRFP (Rocha et al, 2009) and anti‐VPS18 (Abcam #ab178416), as well as mouse anti‐RFP (5F8, Chromotek), anti‐HA (HA.11 (16B12), Covance), anti‐Myc (9E10, Sigma), anti‐GST (2F3) (van Zeijl et al, 2007), anti‐β‐actin (AC‐15, Sigma), anti‐FLAG M2‐PO (Sigma #A8592) and anti‐HA‐PO (Roche #12013819001). The following secondary detection agents were used when applicable: rabbit anti‐mouse‐PO (Dako # P0161), HRP‐Protein A (Invitrogen #10‐1023), IRDye 800CW goat anti‐rabbit IgG (H+L) (Li‐COR #926‐32211), IRDye 800CW goat anti‐mouse IgG (H+L) (Li‐COR #926‐32210), IRDye 680LT goat anti‐rabbit IgG (H+L) (Li‐COR #926‐68021) and IRDye 680LT goat anti‐mouse IgG (H+L) (Li‐COR #926‐68020).

The following antibodies were used for detection
of endogenous protein by Western blot analysis in a 1:1000 dilution:
rabbit anti-PARK7 (Abcam, Cat# ab18257) and rabbit anti-UCHL1(Abcam,
Cat# ab27053). Mouse anti-β-actin (Sigma-Aldrich, Cat# A5441)
was used as a loading control in a 1:10,000 dilution for the Western
blot analysis. Secondary IRDye 800CW goat anti-rabbit IgG (H + L)
(Li-COR, Cat# 926-32211, 1:5000) and IRDye 680LT goat anti-mouse IgG
(H + L) (Li-COR, Cat# 926-68020, 1:20,000) were used for detection
using an Odyssey Classic imager (LI-COR).

The following antibodies
were used for the detection of endogenous protein by immunoblot analysis
in a 1:1000 dilution: rabbit anti-PARK7 (Abcam, Cat# ab18257), rabbit
anti-UCHL1 (Abcam, Cat# ab27053), Mouse anti-β-actin (Sigma-Aldrich,
Cat# A5441) was used as a loading control in a 1:10,000 dilution for
Western blot. Secondary IRDye 800CW goat antirabbit IgG (H + L) (Li-COR,
Cat# 926-32211, 1:5000) and IRDye 680LT goat antimouse IgG (H + L)
(Li-COR, Cat# 926-68020, 1:20,000) were used for detection using the
Odyssey Classic imager (LI-COR).

Mouse tissues were lysed in T-PER reagent with a protease inhibitor cocktail (Thermo Scientific). Equal amounts of proteins were fractionated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes. Blots were probed overnight at 4°C with the following primary antibodies: rabbit anti-TH (1:4000, Sigma), mouse anti-β-actin (1:2000, Abgent), rabbit anti-GFAP (1:4000, Millipore), mouse anti-GAPDH (1:5000, Santa Cruz). Next, the membranes were incubated for 1 h with IRDye 680LT goat anti-mouse IgG (H+L) (926-68020, Li-Cor, USA) or IRDye 800CW goat anti-rabbit IgG (H+L) (926-32211, Li-Cor, USA) (1:10,000 dilutions in TBST with 0.02% SDS) and protein bands detected by an Odyssey infrared imaging system (Li-Cor, USA). Protein levels were quantified by densitometry using Quantity One 4.5.2 software (Bio-Rad, Hercules, USA).